Anti-CD123 antibodies and conjugates and derivatives thereof

ABSTRACT

The present invention generally relates to antibodies, antigen-binding fragments thereof, polypeptides, and immunoconjugates that bind to CD123 antigen (the α chain of the interleukine 3 receptor, or IL-3Rα). The present invention also relates to methods of using such CD123-binding molecules for diagnosing and treating diseases, such as B-cell malignancies.

REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of the filing date, under 35 U.S.C.§ 119(e), of U.S. Provisional Application No. 62/186,161, filed on Jun.29, 2015, U.S. Provisional Application No. 62/338,203, filed on May 18,2016, and U.S. Provisional Application No. 62/346,730, filed on Jun. 7,2016. The entire contents of each of the above-referenced applicationsare incorporated herein by reference.

FIELD OF THE INVENTION

The present invention generally relates to antibodies, antigen-bindingfragments thereof, polypeptides, and immunoconjugates that bind to CD123antigen (the α chain of the interleukin-3 receptor, or IL-3Rα). Thepresent invention also relates to methods of using such CD123-bindingmolecules for diagnosing and treating diseases, such as B-cellmalignancies.

BACKGROUND OF THE INVENTION

CD123 (interleukin 3 receptor alpha, IL-3Rα) is a 40 kDa molecule thatis part of the interleukin 3 receptor (IL-3R) complex. The cytokineInterleukin 3 (IL-3) drives early differentiation of multipotent stemcells into cells of the erythroid, myeloid and lymphoid progenitors.CD123 is expressed on CD34⁺-committed progenitors, but not byCD34⁺/CD38⁻ normal hematopoietic stem cells (HSCs). CD123 is expressedby basophils, mast cells, plasmacytoid dendritic cells, some expressionby monocytes, macrophages and eosinophils, and low or no expression byneutrophils and megakaryocytes. Some non-hematopoietic tissues, such asplacenta, Leydig cells of the testis, certain brain cell elements andsome endothelial cells, also express CD123. However, expression there ismostly cytoplasmic.

CD123 is reported to be expressed by leukemic blast cells (“leukemiablasts”) and leukemia stem cells (LSC) (Jordan et al., Leukemia14:1777-1784, 2000; Jin et al., Blood 113:6603-6610, 2009). In humannormal precursor populations, CD123 is expressed by a subset ofhematopoietic progenitor cells (HPC), but not by normal HSCs. CD123 isalso reportedly expressed by plasmacytoid dendritic cells (pDC) andbasophils, and, to a lesser extent, monocytes and eosinophils.

CD123 has been reported to be overexpressed on malignant cells in a widerange of hematologic malignancies including acute myeloid leukemia (AML)and myelodysplastic syndrome (MDS) (Muñoz et al., Haematologica86(12):1261-1269, 2001). Overexpression of CD123 is associated withpoorer prognosis in AML (Tettamanti et al., Br. J. Haematol.161:389-401, 2013). AML and MDS are thought to arise in and beperpetuated by a small population of leukemic stem cells (LSCs), whichare generally dormant (i.e., not rapidly dividing cells) and thereforeresist cell death (apoptosis) and conventional chemotherapeutic agents.LSCs are characterized by over-expression of CD123, while CD123 is notpresent in the corresponding normal hematopoietic stem cell populationin normal human bone marrow (Jin et al., Blood 113:6603-6610, 2009;Jordan et al., Leukemia 14:1777-1784, 2000). CD123 expression is alsoassociated with multiple other malignancies/pre-malignancies: chronicmyeloid leukemia (CML) progenitor cells (including blast crisis CML);Hodgkin's Reed Sternberg (RS) cells; transformed non-Hodgkin's lymphoma(NHL); some chronic lymphocytic leukemia (CLL) (CD11c⁺); a subset ofacute T lymphoblastic leukemia (T-ALL) (16%, most immature, mostlyadult), plasmacytoid dendritic cell (pDC) (DC2) malignancies andCD34⁺/CD38⁻ myelodysplastic syndrome (MDS) marrow cell malignancies.

AML is a clonal disease characterized by the proliferation andaccumulation of transformed myeloid progenitor cells in the bone marrow,which ultimately leads to hematopoietic failure. The incidence of AMLincreases with age, and older patients typically have worse treatmentoutcomes than do younger patients (Robak et al., Clin. Ther.2:2349-2370, 2009). Unfortunately, at present, most adults with AML diefrom their disease.

Treatment for AML initially focuses in the induction of remission(induction therapy). Once remission is achieved, treatment shifts tofocus on securing such remission (post-remission or consolidationtherapy) and, in some instances, maintenance therapy. The standardremission induction paradigm for AML is chemotherapy with ananthracycline/cytarabine combination, followed by either consolidationchemotherapy, usually with higher doses of the same drugs as were usedduring the induction period, or human stem cell transplantation,depending on the patient's ability to tolerate intensive treatment andthe likelihood of cure with chemotherapy alone (see Roboz, Curr. Opin.Oncol. 24:711-719, 2012).

Agents frequently used in induction therapy include cytarabine and theanthracyclines. Cytarabine, also known as AraC, kills cancer cells andother rapidly dividing normal cells by interfering with DNA synthesis.Side effects associated with AraC treatment include decreased resistanceto infection, a result of decreased white blood cell production;bleeding, as a result of decreased platelet production; and anemia, dueto a potential reduction in red blood cells. Other side effects includenausea and vomiting. Anthracyclines (e.g., daunorubicin, doxorubicin,and idarubicin) have several modes of action including inhibition of DNAand RNA synthesis, disruption of higher order structures of DNA, andproduction of cell damaging free oxygen radicals. The most consequentialadverse effect of anthracyclines is cardiotoxicity, which considerablylimits administered life-time dose and to some extent their usefulness.

Thus, unfortunately, despite substantial progress in the treatment ofnewly diagnosed AML, 20% to 40% of patients do not achieve remissionwith the standard induction chemotherapy, and 50% to 70% of patientsentering a first complete remission are expected to relapse within 3years. The optimum strategy at the time of relapse, or for patients withthe resistant disease, remains uncertain. Stem cell transplantation hasbeen established as the most effective form of antileukemic therapy inpatients with AML in first or subsequent remission (Roboz, 2012).

Antibody-drug conjugates (ADC) and other cell binding agent-drugconjugates are emerging as a powerful class of anti-tumor agents withefficacy across a range of cancers. Cell binding agent-drug conjugates(such as ADCs) are commonly composed of three distinct elements: acell-binding agent (e.g., an antibody); a linker; and a cytotoxicmoiety. Conventionally, the cytotoxic drug moiety is covalently attachedto lysine residues on the antibody, or to cysteine residues, obtainedthrough reduction of interchain disulfide bonds, resulting in ADCs thatare heterogeneous mixtures bearing varying numbers of drugs attached atdifferent positions on the antibody molecule.

SUMMARY OF THE INVENTION

The present invention is based on the surprising findings that theconjugates of the present invention are highly potent against variousCD123-expressing cancer cells, particularly leukemia with at least onenegative prognostic factors.

One aspect of the invention provides an antibody or antigen-bindingfragment thereof that: (a) binds an epitope within amino acids 101 to346 of human CD123/IL3-Rα antigen, and (b) inhibits IL3-dependentproliferation in antigen-positive TF-1 cells.

In certain embodiments, the antibody or antigen-binding fragment thereofbinds an epitope within amino acids 101 to 204 of human CD123 antigen.In another embodiment, the antibody or antigen-binding fragment thereofbinds an epitope within amino acids 205 to 346 of human CD123 antigen.

A related aspect of the invention provides an antibody orantigen-binding fragment thereof that: (a) binds an epitope within aminoacids 1 to 100 of human CD123 antigen, and (b) inhibits IL3-dependentproliferation in antigen-positive TF-1 cells, with an IC₅₀ value of 0.1nM or less (e.g., 0.08 nM, 0.05 nM, 0.03 nM).

In certain embodiments, the antibody or antigen-binding fragment thereofinhibits the proliferation of leukemic stem cells or leukemic blastcells but not hematopoietic stem cells.

In certain embodiments, the antibody or antigen-binding fragment thereofbinds to human CD123 antigen-positive cells with a dissociation constant(K_(d)) of 0.3 nM or lower, such as between 0.01 nM and 0.3 nM, between0.01 nM and 0.2 nM, between 0.01 nM and 0.19 nM, between 0.01 nM and0.18 nM, between 0.01 nM and 0.15 nM, or between 0.01 nM and 0.1 nM.

In certain embodiments, the antibody or antigen-binding fragment thereofbinds to cynomolgus monkey CD123. For example, the antibody orantigen-binding fragment thereof may bind to cynomolgus monkey CD123with a K_(d) of between 0.05 and 0.3 nM, between 0.05 and 0.2 nM,between 0.05 nM and 0.19 nM, between 0.05 nM and 0.18 nM, between 0.05nM and 0.15 nM, or between 0.05 and 0.1 nM. In certain embodiments, theantibody or antigen-binding fragment thereof binds both human andcynomolgus monkey CD123 with a substantially similar binding affinity.For example, the antibody or antigen-binding fragment thereof may bindto human and cynomolgus monkey CD123 with a K_(d) of between 0.05 and0.3 nM, between 0.05 and 0.2 nM, or between 0.05 and 0.1 nM. The K_(d)may be measured by flow cytometry, surface plasmon resonance, orradioimmunoassay.

In certain embodiments, the antibody or antigen-binding fragment thereofinhibits at least 50% of IL3-dependent proliferation in antigen-positiveTF-1 cells at a concentration of 0.5 nM or lower.

In certain embodiments, the antibody or antigen-binding fragment thereofmay comprise: a) at least one heavy chain variable region or fragmentthereof comprising three sequential complementarity-determining regions(CDR) CDR1, CDR2, and CDR3, respectively, wherein, with the exception of1, 2, or 3 conservative amino acid substitutions, CDR1 is selected fromthe group consisting of: SEQ ID NOs: 1, 5, and 12, CDR2 is selected fromthe group consisting of: SEQ ID NOs: 2, 3, 6-10, 13, and 14, and,optionally, CDR3 is selected from the group consisting of: SEQ ID NOs:4, 11, 15 and 70; and b) at least one light chain variable region orfragment thereof comprising three sequential complementarity-determiningregions (CDR) CDR1, CDR2, and CDR3, respectively, wherein, with theexception of 1, 2, or 3 conservative amino acid substitutions, CDR1 isselected from the group consisting of: SEQ ID NOs: 16, 19, 20, 23 and72, CDR2 is selected from the group consisting of: SEQ ID NOs: 17, 21,24 and 71, and, optionally, CDR3 is selected from the group consistingof: SEQ ID NOs: 18, 22, and 25.

In certain embodiments, the antibody or antigen-binding fragment thereofmay comprise: a) at least one heavy chain variable region or fragmentthereof comprising three sequential complementarity-determining regions(CDR) CDR1, CDR2, and CDR3, respectively, wherein, with the exception of1, 2, or 3 conservative amino acid substitutions, CDR1 is selected fromthe group consisting of: SEQ ID NOs: 1, 5, and 12, CDR2 is selected fromthe group consisting of: SEQ ID NOs: 2, 3, 6-10, 13, and 14, and,optionally, CDR3 is selected from the group consisting of: SEQ ID NOs:4, 11 and 15; and b) at least one light chain variable region orfragment thereof comprising three sequential complementarity-determiningregions (CDR) CDR1, CDR2, and CDR3, respectively, wherein, with theexception of 1, 2, or 3 conservative amino acid substitutions, CDR1 isselected from the group consisting of: SEQ ID NOs: 16, 19, 20 and 23,CDR2 is selected from the group consisting of: SEQ ID NOs: 17, 21, and24, and, optionally, CDR3 is selected from the group consisting of: SEQID NOs: 18, 22, and 25.

In certain embodiments, the conservative amino acid substitutionscomprise a substitution of at least one Lys in a CDR by an Arg.

In certain embodiments, the antibody is a CDR-grafted humanized antibodycomprising mouse CDR regions, and wherein one or more (e.g., 1, 2, 3, 4,5, 6, 7, or 8) heavy chain and/or light chain framework region vernierzone residues of said antibody is of mouse origin.

In certain embodiments, the antibody or antigen-binding fragment thereofmay comprise: a) an immunoglobulin heavy chain variable region havingthe amino acid sequence set forth in SEQ ID NO: 39 or 40; and b) animmunoglobulin light chain variable region having the amino acidsequence set forth in SEQ ID NO: 41.

In certain embodiments, the antibody or antigen-binding fragment thereofmay comprise: a) an immunoglobulin heavy chain variable region havingthe amino acid sequence set forth in SEQ ID NO: 34; and b) animmunoglobulin light chain variable region having the amino acidsequence set forth in SEQ ID NO: 35. In certain embodiments, Xaa, thesecond residue from the N-terminus of SEQ ID NO: 34, is Phe. In otherembodiments, Xaa is Val.

In certain embodiments, the antibody or antigen-binding fragment thereofmay comprise: a) an immunoglobulin heavy chain variable region havingthe amino acid sequence set forth in SEQ ID NO: 39 or 40, except thatthe N-terminal residue is Ser; and b) an immunoglobulin light chainvariable region having the amino acid sequence set forth in SEQ ID NO:41.

In certain embodiments, the antibody or antigen-binding fragment thereofmay comprise: a) an immunoglobulin heavy chain variable region havingthe amino acid sequence set forth in SEQ ID NO: 39 or 40; and b) animmunoglobulin light chain variable region having the amino acidsequence set forth in SEQ ID NO: 41, except that the N-terminal residueis Ser.

In certain embodiments, the antibody or antigen-binding fragment thereofmay comprise: a) an immunoglobulin heavy chain region having the aminoacid sequence set forth in SEQ ID NO: 59 or 60, except that theN-terminal residue is Ser, and except that the residue corresponding tothe 5^(th) to the last residue of SEQ ID NO: 54 is Cys (i.e., Cys atEU/OU numbering position 442); and b) an immunoglobulin light chainvariable region having the amino acid sequence set forth in SEQ ID NO:41.

In certain embodiments, the antibody or antigen-binding fragment thereofmay comprise: a) an immunoglobulin heavy chain region having the aminoacid sequence set forth in SEQ ID NO: 59 or 60, except that the residuecorresponding to the 5^(th) to the last residue of SEQ ID NO: 54 is Cys;and b) an immunoglobulin light chain variable region having the aminoacid sequence set forth in SEQ ID NO: 41, except that the N-terminalresidue is Ser.

In certain embodiments, the antibody or antigen-binding fragment thereofmay comprise: a) an immunoglobulin heavy chain variable region havingthe amino acid sequence set forth in SEQ ID NO: 38; and b) animmunoglobulin light chain variable region having the amino acidsequence set forth in SEQ ID NO: 35.

In certain embodiments, the antibody or antigen-binding fragment thereofmay comprise: a) an immunoglobulin heavy chain variable region havingthe amino acid sequence set forth in SEQ ID NO: 34; and b) animmunoglobulin light chain variable region having the amino acidsequence set forth in SEQ ID NO: 37.

In certain embodiments, the antibody or antigen-binding fragment thereofmay comprise: a) an immunoglobulin heavy chain region having the aminoacid sequence set forth in SEQ ID NO: 56; and b) an immunoglobulin lightchain variable region having the amino acid sequence set forth in SEQ IDNO: 35.

In certain embodiments, the antibody or antigen-binding fragment thereofmay comprise: a) an immunoglobulin heavy chain region having the aminoacid sequence set forth in SEQ ID NO: 54; and b) an immunoglobulin lightchain variable region having the amino acid sequence set forth in SEQ IDNO: 37.

In certain embodiments, Xaa, the second residue from the N-terminus ofSEQ ID NOS: 38, 34, 56, and 54, is Phe. In other embodiments, Xaa isVal.

In certain embodiments, the antibody or antigen-binding fragment thereofmay comprise: a) an immunoglobulin heavy chain region having the aminoacid sequence set forth in SEQ ID NO: 59 or 60, except that the residuecorresponding to the 5^(th) to the last residue of SEQ ID NO: 54 is Cys;and b) an immunoglobulin light chain variable region having the aminoacid sequence set forth in SEQ ID NO: 41.

In certain embodiments, the antibody or antigen-binding fragment thereofmay comprise: a) an immunoglobulin heavy chain region having the aminoacid sequence set forth in SEQ ID NO: 54; and b) an immunoglobulin lightchain variable region having the amino acid sequence set forth in SEQ IDNO: 35.

In certain embodiments, Xaa, the second residue from the N-terminus ofSEQ ID NO: 54 or 56, is Phe. In other embodiments, Xaa is Val.

In certain embodiments, the antibody or antigen-binding fragment thereofmay comprise: a) an immunoglobulin heavy chain variable regioncomprising a CDR1 having an amino acid sequence set forth in SEQ ID NO:1, a CDR2 having an amino acid sequence set forth in SEQ ID NO: 2 or 3,and a CDR3 having an amino acid sequence set forth in SEQ ID NO: 4; andb) an immunoglobulin light chain variable region comprising a CDR1having an amino acid sequence set forth in SEQ ID NO: 16, a CDR2 havingan amino acid sequence set forth in SEQ ID NO: 17, and a CDR3 having anamino acid sequence set forth in SEQ ID NO: 18.

In certain embodiments, the antibody or antigen-binding fragment thereofmay comprise: a) an immunoglobulin heavy chain variable regioncomprising a CDR1 having an amino acid sequence set forth in SEQ ID NO:5, a CDR2 having an amino acid sequence set forth in SEQ ID NO: 6, 7, 8,9, or 10, and a CDR3 having an amino acid sequence set forth in SEQ IDNO: 11; and, b) an immunoglobulin light chain variable region comprisinga CDR1 having an amino acid sequence set forth in SEQ ID NO: 19 or 20, aCDR2 having an amino acid sequence set forth in SEQ ID NO: 21, and aCDR3 having an amino acid sequence set forth in SEQ ID NO: 22.

In certain embodiments, the antibody or antigen-binding fragment thereofmay comprise: a) an immunoglobulin heavy chain variable regioncomprising a CDR1 having an amino acid sequence set forth in SEQ ID NO:12, a CDR2 having an amino acid sequence set forth in SEQ ID NO: 13 or14, and a CDR3 having an amino acid sequence set forth in SEQ ID NO: 15;and b) an immunoglobulin light chain variable region comprising a CDR1having an amino acid sequence set forth in SEQ ID NO: 23, a CDR2 havingan amino acid sequence set forth in SEQ ID NO: 24, and a CDR3 having anamino acid sequence set forth in SEQ ID NO: 25.

In certain embodiments, the antibody or antigen-binding fragment thereofmay comprise: a) a V_(H) sequence at least 95% identical to a referenceV_(H) sequence selected from the group consisting of: SEQ ID NOs: 26,28, 30, 32, 34, 38, 39, and 40 (preferably 26, 28, 30, 32, 34, and 38);and/or, b) a V_(L) sequence at least 95% identical to a reference V_(L)sequence selected from the group consisting of: SEQ ID NOs: 27, 29, 31,33, 35, 37, and 41 (preferably 27, 29, 31, 35, and 37). In certainembodiments, the V_(H) sequence is at least 99% identical to one of SEQID NOs: 26, 28, 30, 32, 34, 38, 39, and 40 (preferably 26, 28, 30, 32,34, and 38), and/or wherein the V_(L) sequence is at least 99% identicalto one of SEQ ID NOs: 27, 29, 31, 33, 35, 37, and 41 (preferably 27, 29,31, 35, and 37). In certain embodiments, the antibody or antigen-bindingfragment thereof may comprise: a) a V_(H) sequence selected from thegroup consisting of SEQ ID NOs: 26, 28, 30, 32, 34, 38, 39, and 40(preferably 26, 28, 30, 32, 34, and 38); and/or, b) a V_(L) sequenceselected from the group consisting of SEQ ID NOs: 27, 29, 31, 33, 35,37, and 41 (preferably 27, 29, 31, 35, and 37). In certain embodiments,the antibody or antigen-binding fragment thereof may comprise a V_(H)sequence of SEQ ID NO: 26 and a V_(L) sequence of SEQ ID NO: 27, or aV_(H) sequence of SEQ ID NO: 28 and a V_(L) sequence of SEQ ID NO: 29,or a V_(H) sequence of SEQ ID NO: 30 and a V_(L) sequence of SEQ ID NO:31, or a V_(H) sequence of SEQ ID NO: 34 and a V_(L) sequence of SEQ IDNO: 35.

In certain embodiments, the antibody is a murine, non-human mammal,chimeric, humanized, or human antibody. For example, the humanizedantibody may be a CDR-grafted antibody or resurfaced antibody. Incertain embodiments, the antibody is a full-length antibody. In certainembodiments, the antigen-binding fragment thereof is an Fab, Fab′,F(ab′)₂, F_(d), single chain Fv or scFv, disulfide linked F_(v), V-NARdomain, IgNar, intrabody, IgGΔCH₂, minibody, F(ab′)₃, tetrabody,triabody, diabody, single-domain antibody, DVD-Ig, Fcab, mAb₂, (scFv)₂,or scFv-Fc.

Another aspect of the invention provides a polypeptide comprising theV_(H) and V_(L) sequences of any of the subject antibody orantigen-binding fragment thereof. The polypeptide may be a fusion with aprotein that is not a pseudomonas toxin.

Another aspect of the invention provides a cell producing the antibodyor antigen-binding fragment thereof of the invention, or the polypeptideof the invention.

Another aspect of the invention provides a method of producing theantibody or antigen-binding fragment thereof of the invention, or thepolypeptide of the invention, comprising: (a) culturing the cell of theinvention; and, (b) isolating the antibody, antigen-binding fragmentthereof, or polypeptide from the cultured cell. In certain embodiments,the cell is eukaryotic cell.

Another aspect of the invention provides an immunoconjugate having thefollowing formula:CBA

Cy^(L1))_(W) _(L) ,

-   -   wherein:        -   CBA is an antibody or antigen-binding fragment thereof of            the invention, or the polypeptide thereof of the invention,            that is covalently linked through a lysine residue to            Cy^(L1);        -   W_(L) is an integer from 1 to 20; and        -   Cy^(L1) is represented by the following formula:

-   -   or a pharmaceutically acceptable salt thereof, wherein:        -   the double line            between N and C represents a single bond or a double bond,            provided that when it is a double bond, X is absent and Y is            —H or a (C₁-C₄)alkyl; and    -   when it is a single bond, X is —H or an amine protecting moiety,        and Y is —OH or —SO₃M;        -   W′ is —NR^(e′),        -   R^(e′) is —(CH₂—CH₂—O)_(n)—R^(k);        -   n is an integer from 2 to 6;        -   R^(k) is —H or -Me;        -   R^(x3) is a (C₁-C₆)alkyl;        -   L′ is represented by the following formula:            —NR₅—P—C(═O)—(CR_(a)R_(b))_(m)—C(═O)—  (B1′); or            —NR₅—P—C(═O)—(CR_(a)R_(b))_(m)—S—Z^(s1)—  (B3′);            -   R₅ is —H or a (C₁-C₃)alkyl;            -   P is an amino acid residue or a peptide containing                between 2 to 20 amino acid residues;            -   R_(a) and R_(b), for each occurrence, are each                independently —H, (C₁-C₃)alkyl, or a charged substituent                or an ionizable group Q;            -   m is an integer from 1 to 6; and            -   Z^(s1) is selected from any one of the following                formulas:

-   -   wherein:        -   q is an integer from 1 to 5; and        -   M is H⁺ or a cation.

In certain embodiments, R_(a) and R_(b) are both H; and R₅ is H or Me.

In certain embodiments, P is a peptide containing 2 to 5 amino acidresidues. For example, P may be selected from Gly-Gly-Gly, Ala-Val,Val-Ala, Val-Cit, Val-Lys, Phe-Lys, Lys-Lys, Ala-Lys, Phe-Cit, Leu-Cit,Ile-Cit, Trp, Cit, Phe-Ala, Phe-N⁹-tosyl-Arg, Phe-N⁹-nitro-Arg,Phe-Phe-Lys, D-Phe-Phe-Lys, Gly-Phe-Lys, Leu-Ala-Leu, Ile-Ala-Leu,Val-Ala-Val, Ala-Leu-Ala-Leu (SEQ ID NO: 55), β-Ala-Leu-Ala-Leu (SEQ IDNO: 57), Gly-Phe-Leu-Gly (SEQ ID NO: 73), Val-Arg, Arg-Val, Arg-Arg,Val-D-Cit, Val-D-Lys, Val-D-Arg, D-Val-Cit, D-Val-Lys, D-Val-Arg,D-Val-D-Cit, D-Val-D-Lys, D-Val-D-Arg, D-Arg-D-Arg, Ala-Ala, Ala-D-Ala,D-Ala-Ala, D-Ala-D-Ala, Ala-Met, and Met-Ala. In certain embodiments, Pis Gly-Gly-Gly, Ala-Val, Ala-Ala, Ala-D-Ala, D-Ala-Ala, or D-Ala-D-Ala.

In certain embodiments, Q is —SO₃M.

In certain embodiments, the immunoconjugate is represented by thefollowing formula:

-   -   or a pharmaceutically acceptable salt thereof, wherein W_(L) is        an integer from 1 to 10; the double line        between N and C represents a single bond or a double bond,        provided that when it is a double bond, X is absent and Y is —H;        and when it is a single bond, X is —H, and Y is —OH or —SO₃M.    -   A related aspect provides an immunoconjugate having the formula:        CBA        Cy^(L2))_(W) _(L) ,    -   wherein:        -   CBA is an antibody or antigen-binding fragment thereof of            the invention, or the polypeptide thereof of the invention,            that is covalently linked to Cy^(L2) through a lysine            residue;        -   W_(L) is an integer from 1 to 20; and        -   Cy^(L2) is represented by the following formula:

-   -   or a pharmaceutically acceptable salt thereof, wherein:        -   the double line            between N and C represents a single bond or a double bond,            provided that when it is a double bond, X is absent and Y is            —H or a (C₁-C₄)alkyl; and    -   when it is a single bond, X is —H or an amine protecting moiety,        and Y is —OH or —SO₃M;        -   R^(x1) and R^(x2) are independently (C₁-C₆)alkyl;        -   R^(e) is —H or a (C₁-C₆)alkyl;        -   W′ is —NR^(e′),        -   R^(e′) is —(CH₂—CH₂—O)_(n)—R^(k);        -   n is an integer from 2 to 6;        -   R^(k) is —H or -Me;        -   Z^(s1) is selected from any one of the following formulas:

-   -   wherein:        -   q is an integer from 1 to 5; and        -   M is —H⁺ or a cation.

In certain embodiments, R^(e) is H or Me; R^(x1) and R^(x2) areindependently wherein R^(f) and R^(g) are each independently —H or a(C₁-C₄)alkyl; and p is 0, 1, 2 or 3.

In certain embodiments, R^(f) and R^(g) are the same or different, andare selected from —H and -Me.

In certain embodiments, the immunoconjugate is represented by thefollowing formula:

-   -   or a pharmaceutically acceptable salt thereof, wherein W_(L) is        an integer from 1 to 10; the double line        between N and C represents a single bond or a double bond,        provided that when it is a double bond, X is absent and Y is —H;        and when it is a single bond, X is —H and Y is —OH or —SO₃M.

In certain embodiments, the double line

between N and C represents a double bond, X is absent and Y is —H. Incertain embodiments, the double line

between N and C represents a single bond, X is —H, and Y is —SO₃M. Incertain embodiments, M is H⁺, Na⁺ or K⁺.

Another related aspect of the invention provides an immunoconjugatehaving the formula:CBA

Cy^(L3))_(W) _(L) ,

-   -   wherein:        -   CBA is an antibody or antigen-binding fragment thereof of            the invention, or the polypeptide of the invention, which is            covalently linked to Cy^(L3) through a Lys residue;        -   W_(L) is an integer from 1 to 20;        -   Cy^(L3) is represented by the following formula:

-   -   -   m′ is 1 or 2;        -   R₁ and R₂, are each independently H or a (C₁-C₃)alkyl; and        -   Z^(s1) is selected from any one of the following formulas:

-   -   wherein:        -   q is an integer from 1 to 5; and        -   M is H⁺ or a cation.

In certain embodiments, m′ is 1, and R₁ and R₂ are both H. In certainother embodiments, m′ is 2, and R₁ and R₂ are both Me.

In certain embodiments, the immunoconjugate is represented by thefollowing formula:

-   -   or a pharmaceutically acceptable salt thereof, wherein W_(L) is        an integer from 1 to 10.

In certain embodiments, M is H⁺, Na⁺ or K⁺.

Another aspect of the invention provides an immunoconjugate having thefollowing formula:

-   -   wherein:        -   CBA is an antibody or antigen-binding fragment thereof of            the invention, or the polypeptide thereof of the invention,            covalently linked to the J_(CB)′ group;        -   W_(S) is 1, 2, 3, or 4;        -   J_(CB)′ is a moiety formed by reacting an aldehyde group            derived from oxidation of a 2-hydroxyethylamine moiety            (wherein the 2-hydroxyethylamine moiety can be part of a            serine, threonine, hydroxylysine, 4-hydroxyornithie or            2,4-diamino-5-hydroxy valeric acid residue) on an N-terminal            of said antibody or antigen-binding fragment thereof of the            invention, or the polypeptide thereof of the invention, and            an aldehyde reactive group on Cy^(s1), and is represented by            the following formula:

-   -   wherein s1 is the site covalently linked to the CBA; and s2 is        the site covalently linked to Cy^(s1);        -   Cy^(s1) is represented by the following formula:

-   -   or a pharmaceutically acceptable salt thereof, wherein:        -   the double line            between N and C represents a single bond or a double bond,            provided that when it is a double bond, X is absent and Y is            —H or a (C₁-C₄)alkyl; and    -   when it is a single bond, X is —H or an amine protecting moiety,        Y is —OH or —SO₃M, and M is H⁺ or a cation;        -   R₅ is —H or a (C₁-C₃)alkyl;        -   P is an amino acid residue or a peptide containing 2 to 20            amino acid residues;        -   Z_(d1) is absent, —C(═O)—NR₉—, or —NR₉—C(═O)—;        -   R₉ is —H or a (C₁-C₃)alkyl;        -   R_(a) and R_(b), for each occurrence, are independently —H,            (C₁-C₃)alkyl, or a charged substituent or an ionizable group            Q;        -   r and r′ are independently an integer from 1 to 6;        -   W′ is —NR^(e′),        -   R^(e′) is —(CH₂—CH₂—O)_(n)—R^(k);        -   n is an integer from 2 to 6;        -   R^(k) is —H or -Me;        -   R^(x3) is a (C₁-C₆)alkyl;        -   L is —NR₉—(CR_(a)R_(b))_(r″) or absent; and        -   r″ is an integer from 0 to 6.

For simplicity, in each instance below reciting Ser as the N-terminalresidue, it should be understood that other 2-hydroxyethylamine moiety,as part of a serine, threonine, hydroxylysine, 4-hydroxyornithie or2,4-diamino-5-hydroxy valeric acid residue, is contemplated whereapplicable, especially with respect to Thr.

In certain embodiments, R_(a) and R_(b) are both H, and R₅ and R₉ areboth H or Me.

In certain embodiments, P is a peptide containing 2 to 5 amino acidresidues. For example, P may be selected from the group consisting of:Gly-Gly-Gly, Ala-Val, Val-Ala, Val-Cit, Val-Lys, Phe-Lys, Lys-Lys,Ala-Lys, Phe-Cit, Leu-Cit, Ile-Cit, Trp, Cit, Phe-Ala, Phe-N⁹-tosyl-Arg,Phe-N⁹-nitro-Arg, Phe-Phe-Lys, D-Phe-Phe-Lys, Gly-Phe-Lys, Leu-Ala-Leu,Ile-Ala-Leu, Val-Ala-Val, Ala-Leu-Ala-Leu (SEQ ID NO: 55),β-Ala-Leu-Ala-Leu (SEQ ID NO: 57), Gly-Phe-Leu-Gly (SEQ ID NO: 73),Val-Arg, Arg-Val, Arg-Arg, Val-D-Cit, Val-D-Lys, Val-D-Arg, D-Val-Cit,D-Val-Lys, D-Val-Arg, D-Val-D-Cit, D-Val-D-Lys, D-Val-D-Arg,D-Arg-D-Arg, Ala-Ala, Ala-D-Ala, D-Ala-Ala, D-Ala-D-Ala, Ala-Met, andMet-Ala. In certain embodiments, P is Gly-Gly-Gly, Ala-Val, Ala-Ala,Ala-D-Ala, D-Ala-Ala, or D-Ala-D-Ala. In certain embodiments, Q is—SO₃M.

In certain embodiments, the immunoconjugate is represented by thefollowing formula:

-   -   or a pharmaceutically acceptable salt thereof, wherein the        double line        between N and C represents a single bond or a double bond,        provided that when it is a double bond, X is absent and Y is —H,        and when it is a single bond, X is —H, and Y is —OH or —SO₃M.

Another aspect of the invention provides an immunoconjugate having thefollowing formula:

-   -   wherein:        -   CBA is the antibody or antigen-binding fragment thereof of            the invention, or the polypeptide thereof of the invention,            covalently linked to the J_(CB)′ group,        -   J_(CB)′ is a moiety formed by reacting an aldehyde group            derived from oxidation of a 2-hydroxyethylamine moiety on an            N-terminal of said antibody or antigen-binding fragment            thereof of the invention, or the polypeptide thereof of the            invention, and an aldehyde reactive group on Cy^(s2), and is            represented by the following formula:

-   -   wherein s1 is the site covalently linked to the CBA; and s2 is        the site covalently linked to Cy^(s2);        -   Cy^(s2) is represented by the following formula:

-   -   or a pharmaceutically acceptable salt thereof, wherein:        -   the double line            between N and C represents a single bond or a double bond,            provided that when it is a double bond, X is absent and Y is            —H or a (C₁-C₄)alkyl; and    -   when it is a single bond, X is —H or an amine protecting moiety,        and Y is —OH or —SO₃M;        -   M is H⁺ or a cation;        -   R^(x1) is a (C₁-C₆)alkyl;        -   R^(e) is —H or a (C₁-C₆)alkyl;        -   W′ is —NR^(e′),        -   R^(e′) is —(CH₂—CH₂—O)_(n)—R^(k);        -   n is an integer from 2 to 6;        -   R^(k) is —H or -Me;        -   R^(x2) is a (C₁-C₆)alkyl;        -   L₁ is represented by the following formula:

-   -   wherein:        -   s3 is the site covalently linked to the group J_(CB)′;        -   s4 is the site covalently linked to the —S— group on            Cy^(s2);        -   Z_(a2) is absent, —C(═O)—NR₉—, or —NR₉—C(═O)—;        -   R₉ is —H or a (C₁-C₃)alkyl;        -   Q is H, a charged substituent or an ionizable group;        -   R_(a1), R_(a2), R_(a3), R_(a4), for each occurrence, are            independently H or (C₁-C₃)alkyl; and        -   q1 and r1 are each independently an integer from 0 to 10,            provided that q1 and r1 are not both 0.

In certain embodiments, -L₁- is represented by the following formula:

-   -   or a pharmaceutically acceptable salt thereof, wherein R is H or        —SO₃M.

In certain embodiments, R^(e) is H or Me; and R^(x1) is—(CH₂)_(p)—(CR^(f)R^(g))—, and R^(x2) is —(CH₂)_(p)—(CR^(f)R^(g))—,wherein R^(f) and R^(g) are each independently —H or a (C₁-C₄)alkyl; andp is 0, 1, 2 or 3. In certain embodiments, R^(f) and R^(g) are the sameor different, and are selected from —H and -Me.

In certain embodiments, the immunoconjugate is represented by thefollowing formula:

-   -   or a pharmaceutically acceptable salt thereof, wherein the        double line        between N and C represents a single bond or a double bond,        provided that when it is a double bond, X is absent and Y is —H;        and when it is a single bond, X is —H; and Y is —OH or —SO₃M.

In certain embodiments, the double line

between N and C represents a double bond, X is absent and Y is —H.

In certain embodiments, the double line

between N and C represents a single bond, X is —H and Y is —SO₃M. Incertain embodiments, M is H⁺, Na⁺ or K⁺.

Another aspect of the invention provides an immunoconjugate having thefollowing formula:

-   -   wherein:        -   CBA is the antibody or antigen-binding fragment thereof of            the invention, or the polypeptide thereof of the invention,            covalently linked to the J_(CB)′ group;        -   J_(CB)′ is a moiety formed by reacting an aldehyde group            derived from oxidation of a 2-hydroxyethylamine moiety on an            N-terminal of said antibody or antigen-binding fragment            thereof of the invention, or the polypeptide thereof of the            invention, and an aldehyde reactive group on Cy^(s3), and is            represented by the following formula:

-   -   wherein s1 is the site covalently linked to the CBA; and s2 is        the site covalently linked to Cy^(s3);        -   Cy^(s3) is represented by the following formula:

-   -   wherein:        -   m′ is 1 or 2;        -   R₁ and R₂, are each independently H or a (C₁-C₃)alkyl;        -   L₁ is represented by the following formula:

-   -   wherein:        -   s3 is the site covalently linked to the group J_(CB)′;        -   s4 is the site covalently linked to the —S— group on            Cy^(s3);        -   Z_(a2) is absent, —C(═O)—NR₉—, or —NR₉—C(═O)—;        -   R₉ is —H or a (C₁-C₃)alkyl;        -   Q is H, a charged substituent or an ionizable group;        -   R_(a1), R_(a2), R_(a3), R_(a4), for each occurrence, are            independently H or a (C₁-C₃)alkyl; and        -   q1 and r1 are each independently an integer from 0 to 10,            provided that q1 and r1 are not both 0.

In certain embodiments, m′ is 1 and R₁ and R₂ are both H. In certainembodiments, m′ is 2 and R₁ and R₂ are both Me.

In certain embodiments, -L₁- is represented by the following formula:

-   -   or a pharmaceutically acceptable salt thereof, wherein R is H or        —SO₃M and M is H⁺ or a cation.

In certain embodiments, the immunoconjugate is represented by thefollowing formula:

-   -   or a pharmaceutically acceptable salt thereof; wherein DM is        represented by the following formula:

Another aspect of the invention provides an immunoconjugate having thefollowing formula:

-   -   wherein:        -   CBA is the antibody or antigen-binding fragment thereof of            the invention, or the polypeptide thereof of the invention,            covalently linked to the J_(CB)′ group,        -   J_(CB)′ is a moiety formed by reacting an aldehyde group            derived from oxidation of a 2-hydroxyethylamine moiety on an            N-terminal of said antibody or antigen-binding fragment            thereof of the invention, or the polypeptide thereof of the            invention, and an aldehyde reactive group on Cy^(s4) and is            represented by the following formula:

-   -   wherein s1 is the site covalently linked to the CBA; and s2 is        the site covalently linked to Cy^(s4);        -   Cy^(s4) is represented by the following formula:

-   -   -   L₁′ is represented by the following formula:

-   -   wherein:        -   s3 is the site covalently linked to the group J_(CB)′ group;        -   s4 is the site covalently linked to —NMe- group on Cy^(s4);        -   Z_(b1) and Z_(b2) are both absent, or one of Z_(b1) and            Z_(b2) is absent and the other is —CH₂—O— or —O—CH₂—;        -   Z_(b1)′ and Z_(b2)′ are each independently absent, —CH₂—O—,            —O—CH₂—, —NR₉—C(═O)—CH₂—, or —CH₂—C(═O)—NR₉—;        -   R₉ is H or (C₁-C₃)alkyl;        -   n1 and m1 are each independently an integer from 1 to 6;        -   one of E₁ and E₂ is —C(═O)—, and the other is —NR₉—; or one            of E₁ and E₂ is —C(═O)— or —NR₉—, and the other is absent;        -   P is an amino acid residue or a peptide containing between 2            to 20 amino acid residues; and        -   R_(b1), R_(b2), R_(b3), R_(b4), R_(b5) and R_(b6), for each            occurrence, are each independently H or a (C₁-C₃)alkyl.

In certain embodiments, R_(b1), R_(b2), R_(b3), R_(b4), R_(b5), andR_(b6) are all H. In certain embodiments, R₉ is H.

In certain embodiments, Z_(b1)′ and Z_(b2)′ are both absent; or Z_(b1)′is —CH₂—O—; and Z_(b2)′ is absent; or Z_(b1)′ is —CH₂—C(═O)—NR₉—; andZ_(b2)′ is —O—CH₂— or absent.

In certain embodiments, P is a peptide containing 2 to 5 amino acidresidues. For example, P may be selected from Gly-Gly-Gly, Ala-Val,Val-Ala, Val-Cit, Val-Lys, Phe-Lys, Lys-Lys, Ala-Lys, Phe-Cit, Leu-Cit,Ile-Cit, Trp, Cit, Phe-Ala, Phe-N⁹-tosyl-Arg, Phe-N⁹-nitro-Arg,Phe-Phe-Lys, D-Phe-Phe-Lys, Gly-Phe-Lys, Leu-Ala-Leu, Ile-Ala-Leu,Val-Ala-Val, Ala-Leu-Ala-Leu (SEQ ID NO: 55), β-Ala-Leu-Ala-Leu (SEQ IDNO: 57), Gly-Phe-Leu-Gly (SEQ ID NO: 73), Val-Arg, Arg-Val, Arg-Arg,Val-D-Cit, Val-D-Lys, Val-D-Arg, D-Val-Cit, D-Val-Lys, D-Val-Arg,D-Val-D-Cit, D-Val-D-Lys, D-Val-D-Arg, D-Arg-D-Arg, Ala-Ala, Ala-D-Ala,D-Ala-Ala, D-Ala-D-Ala, Ala-Met, and Met-Ala. In certain embodiments, Pis Gly-Gly-Gly, Ala-Val, Ala-Ala, Ala-D-Ala, D-Ala-Ala, and D-Ala-D-Ala.

In certain embodiments, immunoconjugate is represented by the followingformula:

-   -   or a pharmaceutically acceptable salt thereof, wherein DM is        represented by the following structural formula:

Another aspect of the invention provides an immunoconjugate representedby the following formula:CBA

Cy^(C1))_(W) _(C) ,

-   -   wherein:        -   CBA is an antibody or antigen-binding fragment thereof of            the invention, or the polypeptide thereof of the invention,            covalently linked to Cy^(C1) through a cysteine residue;        -   W_(C) is 1 or 2;        -   Cy^(C1) is represented by the following formula:

-   -   or a pharmaceutically acceptable salt thereof, wherein:    -   the double line        between N and C represents a single bond or a double bond,        provided that when it is a double bond, X is absent and Y is —H        or a (C₁-C₄)alkyl; and    -   when it is a single bond, X is —H or an amine protecting moiety,        Y is —OH or —SO₃M, and M is H⁺ or a cation;    -   R₅ is —H or a (C₁-C₃)alkyl;    -   P is an amino acid residue or a peptide containing 2 to 20 amino        acid residues;    -   R_(a) and R_(b), for each occurrence, are independently —H,        (C₁-C₃)alkyl, or a charged substituent or an ionizable group Q;    -   W′ is —NR^(e′),    -   R^(e′) is —(CH₂—CH₂—O)_(n)—R^(k);    -   n is an integer from 2 to 6;    -   R^(k) is —H or -Me;    -   R^(x3) is a (C₁-C₆)alkyl; and,    -   L_(C) is represented by

s1 is the site covalently linked to CBA, and s2 is the site covalentlylinked to the —C(═O)— group on Cy^(C1); wherein:

-   -   R₁₉ and R₂₀, for each occurrence, are independently —H or a        (C₁-C₃)alkyl;    -   m″ is an integer between 1 and 10; and    -   R^(h) is —H or a (C₁-C₃)alkyl.

In certain embodiments, R_(a) and R_(b) are both H; and R₅ is H or Me.

In certain embodiments, P is a peptide containing 2 to 5 amino acidresidues. For example, P may be selected from Gly-Gly-Gly, Ala-Val,Val-Ala, Val-Cit, Val-Lys, Phe-Lys, Lys-Lys, Ala-Lys, Phe-Cit, Leu-Cit,Ile-Cit, Trp, Cit, Phe-Ala, Phe-N⁹-tosyl-Arg, Phe-N⁹-nitro-Arg,Phe-Phe-Lys, D-Phe-Phe-Lys, Gly-Phe-Lys, Leu-Ala-Leu, Ile-Ala-Leu,Val-Ala-Val, Ala-Leu-Ala-Leu (SEQ ID NO: 55), β-Ala-Leu-Ala-Leu (SEQ IDNO: 57), Gly-Phe-Leu-Gly (SEQ ID NO: 73), Val-Arg, Arg-Val, Arg-Arg,Val-D-Cit, Val-D-Lys, Val-D-Arg, D-Val-Cit, D-Val-Lys, D-Val-Arg,D-Val-D-Cit, D-Val-D-Lys, D-Val-D-Arg, D-Arg-D-Arg, Ala-Ala, Ala-D-Ala,D-Ala-Ala, D-Ala-D-Ala, Ala-Met, and Met-Ala. In certain embodiments, Pis Gly-Gly-Gly, Ala-Val, Ala-Ala, Ala-D-Ala, D-Ala-Ala, or D-Ala-D-Ala.In certain embodiments, Q is —SO₃M.

In certain embodiments, R₁₉ and R₂₀ are both H; and m″ is an integerfrom 1 to 6.

In certain embodiments, -L_(C)- is represented by the following formula:

In certain embodiments, the immunoconjugate is represented by thefollowing formula:

-   -   or a pharmaceutically acceptable salt thereof, wherein the        double line        between N and C represents a single bond or a double bond,        provided that when it is a double bond, X is absent and Y is —H,        and when it is a single bond, X is —H, and Y is —OH or —SO₃M.

Another aspect of the invention provides an immunoconjugate representedby the following formula:CBA

Cy^(C2))_(W) _(C) ,

-   -   wherein:        -   CBA is an antibody or antigen-binding fragment thereof of            the invention, or the polypeptide thereof of the invention,            covalently linked to Cy^(C2) through a cysteine residue;        -   W_(C) is 1 or 2;        -   Cy^(C2) is represented by the following formula:

-   -   or a pharmaceutically acceptable salt thereof, wherein:        -   the double line            between N and C represents a single bond or a double bond,            provided that when it is a double bond, X is absent and Y is            —H or a (C₁-C₄)alkyl; and    -   when it is a single bond, X is —H or an amine protecting moiety,        Y is —OH or —SO₃M, and M is H⁺ or a cation;        -   R^(x1) is a (C₁-C₆)alkyl;        -   R^(e) is —H or a (C₁-C₆)alkyl;        -   W′ is —NR^(e′);        -   R^(e′) is —(CH₂—CH₂—O)_(n)—R^(k);        -   n is an integer from 2 to 6;        -   R^(k) is —H or -Me;        -   R^(x2) is a (C₁-C₆)alkyl;        -   L_(C)′ is represented by the following formula:

-   -   wherein:        -   s1 is the site covalently linked to the CBA and s2 is the            site covalently linked to —S— group on Cy^(C2);        -   Z is —C(═O)—NR₉—, or —NR₉—C(═O)—;        -   Q is —H, a charged substituent, or an ionizable group;        -   R₉, R₁₀, R₁₁, R₁₂, R₁₃, R₁₉, R₂₀, R₂₁ and R₂₂, for each            occurrence, are independently —H or a (C₁-C₃)alkyl;        -   q and r, for each occurrence, are independently an integer            between 0 and 10;        -   m and n are each independently an integer between 0 and 10;        -   R^(h) is —H or a (C₁-C₃)alkyl; and        -   P′ is an amino acid residue or a peptide containing 2 to 20            amino acid residues.

In certain embodiments, P′ is a peptide containing 2 to 5 amino acidresidues. For example, P′ may be selected from Gly-Gly-Gly, Ala-Val,Val-Ala, Val-Cit, Val-Lys, Phe-Lys, Lys-Lys, Ala-Lys, Phe-Cit, Leu-Cit,Ile-Cit, Trp, Cit, Phe-Ala, Phe-N⁹-tosyl-Arg, Phe-N⁹-nitro-Arg,Phe-Phe-Lys, D-Phe-Phe-Lys, Gly-Phe-Lys, Leu-Ala-Leu, Ile-Ala-Leu,Val-Ala-Val, Ala-Leu-Ala-Leu (SEQ ID NO: 55), β-Ala-Leu-Ala-Leu (SEQ IDNO: 57), Gly-Phe-Leu-Gly (SEQ ID NO: 73), Val-Arg, Arg-Val, Arg-Arg,Val-D-Cit, Val-D-Lys, Val-D-Arg, D-Val-Cit, D-Val-Lys, D-Val-Arg,D-Val-D-Cit, D-Val-D-Lys, D-Val-D-Arg, D-Arg-D-Arg, Ala-Ala, Ala-D-Ala,D-Ala-Ala, D-Ala-D-Ala, Ala-Met, and Met-Ala. In certain embodiments, P′is Gly-Gly-Gly, Ala-Val, Ala-Ala, Ala-D-Ala, D-Ala-Ala, or D-Ala-D-Ala.

In certain embodiments, in the immunoconjugate -L_(C)′- is representedby the following formula:

In certain embodiments, R^(e) is H or Me; R^(x1) is—(CH₂)_(p)—(CR^(f)R^(g))—, and R^(x2) is —(CH₂)_(p)—(CR^(f)R^(g))—,wherein R^(f) and R^(g) are each independently —H or a (C₁-C₄)alkyl; andp is 0, 1, 2 or 3. In certain embodiments, R^(f) and R^(g) are the sameor different, and are selected from —H and -Me.

In certain embodiments, the immunoconjugate is represented by thefollowing formula:

-   -   or a pharmaceutically acceptable salt thereof, wherein the        double line        between N and C represents a single bond or a double bond,        provided that when it is a double bond, X is absent and Y is —H,        and when it is a single bond, X is —H, and Y is —OH or —SO₃M.

In certain embodiments, the double line

between N and C represents a double bond, X is absent and Y is —H.

In certain embodiments, the double line

between N and C represents a single bond, X is —H and Y is —SO₃M. Incertain embodiments, M is H⁺, Na⁺ or K⁺.

Another aspect of the invention provides an immunoconjugate having thefollowing formula:CBA

Cy^(C3))_(W) _(C) ,

-   -   wherein:        -   CBA is an antibody or antigen-binding fragment thereof of            the invention, or the polypeptide thereof of the invention,            covalently linked to Cy^(C3) through a cysteine residue;        -   W_(C) is 1 or 2;        -   Cy^(C3) is represented by the following formula:

-   -   wherein:        -   m′ is 1 or 2;        -   R₁ and R₂, are each independently —H or a (C₁-C₃)alkyl;        -   L_(C)′ is represented by the following formula:

-   -   wherein:        -   s1 is the site covalently linked to the CBA and s2 is the            site covalently linked to —S— group on Cy^(C3);        -   Z is —C(═O)—NR₉—, or —NR₉—C(═O)—;        -   Q is H, a charged substituent, or an ionizable group;        -   R₉, R₁₀, R₁₁, R₁₂, R₁₃, R₁₉, R₂₀, R₂₁ and R₂₂, for each            occurrence, are independently —H or a (C₁-C₃)alkyl;        -   q and r, for each occurrence, are independently an integer            between 0 and 10;        -   m and n are each independently an integer between 0 and 10;        -   R^(h) is —H or a (C₁-C₃)alkyl; and        -   P′ is an amino acid residue or a peptide containing 2 to 20            amino acid residues.

In certain embodiments, P′ is a peptide containing 2 to 5 amino acidresidues. For example, P′ is selected from Gly-Gly-Gly, Ala-Val,Val-Ala, Val-Cit, Val-Lys, Phe-Lys, Lys-Lys, Ala-Lys, Phe-Cit, Leu-Cit,Lle-Cit, Trp, Cit, Phe-Ala, Phe-N⁹-tosyl-Arg, Phe-N⁹-nitro-Arg,Phe-Phe-Lys, D-Phe-Phe-Lys, Gly-Phe-Lys, Leu-Ala-Leu, Ile-Ala-Leu,Val-Ala-Val, Ala-Leu-Ala-Leu (SEQ ID NO: 55), β-Ala-Leu-Ala-Leu (SEQ IDNO: 57), Gly-Phe-Leu-Gly (SEQ ID NO: 73), Val-Arg, Arg-Val, Arg-Arg,Val-D-Cit, Val-D-Lys, Val-D-Arg, D-Val-Cit, D-Val-Lys, D-Val-Arg,D-Val-D-Cit, D-Val-D-Lys, D-Val-D-Arg, D-Arg-D-Arg, Ala-Ala, Ala-D-Ala,D-Ala-Ala, D-Ala-D-Ala, Ala-Met, and Met-Ala. In certain embodiments, P′is Gly-Gly-Gly, Ala-Val, Ala-Ala, Ala-D-Ala, D-Ala-Ala, or D-Ala-D-Ala.

In certain embodiments, -L_(C)′- is represented by the followingformula:

-   -   wherein M is H⁺ or a cation.

In certain embodiments, m′ is 1 and R₁ and R₂ are both H. In certainembodiments, m′ is 2 and R₁ and R₂ are both Me.

In certain embodiments, the immunoconjugate is represented by thefollowing formula:

-   -   or a pharmaceutically acceptable salt thereof, wherein DM is a        drug moiety represented by the following formula:

Another aspect of the invention provides a pharmaceutical compositioncomprising the antibody or antigen-binding fragment thereof of theinvention, or the polypeptide of the invention, or the immunoconjugateof the invention, and a pharmaceutically acceptable carrier.

Another aspect of the invention provides a method for inhibiting thegrowth of a cell expressing CD123, comprising contacting the cell withthe antibody or antigen-binding fragment thereof of the invention, orthe polypeptide of the invention, or the immunoconjugate of theinvention, or the pharmaceutical composition of the invention.

In certain embodiments, the cell is a tumor cell. In certainembodiments, the cell is a leukemia cell or a lymphoma cell.

Another aspect of the invention provides a method for treating a subjecthaving cancer, wherein cells of the cancer expresses CD123, the methodcomprising administering to said subject a therapeutically effectiveamount of the antibody or antigen-binding fragment thereof of theinvention, or the polypeptide of the invention, or the immunoconjugateof the invention, or the pharmaceutical composition of the invention.

In certain embodiments, the cancer or cell-proliferative disorder isleukemia or lymphoma. In certain embodiments, the cancer orcell-proliferative disorder is selected from the group consisting of:acute myeloid leukemia (AML); chronic myeloid leukemia (CML); acutelymphoblastic leukemia (ALL), including B-cell lineage acutelymphoblastic leukemia (B-ALL); chronic lymphocytic leukemia (CLL);hairy cell leukemia (HCL); myelodysplastic syndrome; basic plasmacytoidDC neoplasm (BPDCN) leukemia; non-Hodgkin lymphomas (NHL), includingmantle cell lymphoma; and Hodgkin's leukemia (HL). In certainembodiments, the cancer is acute myeloid leukemia (AML). In certainembodiments, the cancer is B-cell acute lymphoblastic leukemia (B-ALL).

Another aspect of the invention provides a method for treating acell-proliferative disorder in a subject, wherein cells of thecell-proliferative disorder expresses CD123, the method comprisingadministering to said subject a therapeutically effective amount of theantibody or antigen-binding fragment thereof of the invention, or thepolypeptide of the invention, or the immunoconjugate of the invention,or the pharmaceutical composition of the invention, in an amountsufficient to treat said cell-proliferative disorder.

It is contemplated that any one embodiment described herein, includingthose described only in one aspect of the invention (but not in othersor not repeated in others), and those described only in the Examples,can be combined with any one or more other embodiments of the invention,unless explicitly disclaimed or inapplicable.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows inhibition of IL-3-dependent proliferation of TF-1 cells bychimeric CD123-6 antibody (chCD123-6) and its CDR-grafted huCD123-6antibodies (huCD123-6Gv4.6 and huCD123-6Gv4.7).

FIGS. 2A and 2B show that three murine anti-CD123 antibodies (muCD123-3,-6 and -14) inhibit IL-3 dependent proliferation of TF-1 cells at leastas well as 7G3. FIG. 2A shows inhibition of TF-1 cells cultured in thepresence of IL-3 (1 ng/mL) by the various anti-CD123 antibodies,including CD123-binding control antibodies 7G3, 6H6, and 9F5. FIG. 2Bshows inhibition of TF-1 cells cultured in the presence of GM-CSF (2ng/mL) by the same anti-CD123 antibodies.

FIG. 3 shows that murine anti-CD123 antibodies muCD123-3, -6, and -14inhibit IL-3 (1 ng/mL) dependent proliferation of TF-1 cells in adose-dependent manner, and to a higher degree than the 7G3 antibody.muCD123-16 is a negative control anti-CD123 antibody that binds CD123but does not inhibit IL-3-dependent proliferation of TF-1 cells.

FIGS. 4A and 4B show that the murine muCD123-3, -6, and -14 antibodieshave higher binding affinity to CD123-positive AML cells than that ofthe 7G3 antibody in CD123-expressing TF-1 (FIG. 4A) and HNT-34 (FIG. 4B)cells.

FIGS. 5A and 5B show that chimeric anti-CD-123 antibodies chCD123-3, -6,and -14 retain high binding affinity of their murine counterparts, usingeither HNT-34 cells (FIG. 5A) or the CD123-positive acute myeloidleukemia (AML) cell line MOLM-13 (FIG. 5B). Chimeric antibody chKTI,which does not bind CD123, was included as negative control.

FIG. 6 shows that the chimeric chCD123-3, -6, and -14 anti-CD-123antibodies retain functional activity of their murine counterparts, asevidenced by their ability to inhibit IL-3 dependent proliferation ofTF-1 cells. A non-functional chimeric anti-CD123 antibody (chCD123-18)that binds CD123 but does not inhibit IL-3-dependent proliferation ofTF-1 cells was included as negative control.

FIG. 7A shows that the murine (muCD123-6), chimeric (chCD123-6), andCDR-grafted huCD123-6 antibodies (huCD123-6Gv4.7S2 and huCD123-6Gv4.7S3)all have higher affinity than 7G3 to CD123-expressing HNT-34 cells.Chimeric antibody chKTI, which does not bind CD123, was included asnegative control. FIGS. 7B and 7C show that conjugation of thehuCD123-6Gv4.7S3 or the -Gv4.7 antibody to the D1 or D2 compoundsthrough Lys-, Ser-, or Cys-linkage only moderately affected the bindingaffinities of these ADC conjugates, i.e., the Ser-linkedhuCD123-6Gv4.7S3-SeriMab-sD1 (see structure in FIG. 17) andhuCD123-6Gv4.7S3-SeriMab-D8, and the Lys-linkedhuCD123-6Gv4.7S3-sSPDB-D1, and huCD123-6Gv4.7S3-D2 in FIG. 7B; and theCys-linked huCD123-6Gv4.7-CysMab-D4 and huCD123-6Gv4.7-CysMab-D5 in FIG.7C. In FIG. 7C, the unconjugated huCD123-6Gv4.7 antibody has a heavychain sequence of SEQ ID NO: 54, in which Xaa is Val. The conjugateswith “53-SeriMab” have cytotoxin (in this case, theindolinobenzodiazepine or “IGN” compounds herein after) linkage throughoxidized N-terminal Ser on light chain. The conjugates with “CysMab”have cytotoxin (in this case, the IGN compounds) linkage through theengineered Cys in the heavy chain (i.e., the Cys corresponding to the5^(th) to the last Cys in SEQ ID NO: 54).

FIG. 8A shows that the chimeric (chCD123-6) and CDR-grafted(huCD123-6Gv4.7S2 and huCD123-6Gv4.7S3) huCD123-6 antibodies inhibitIL-3 dependent proliferation of TF-1 cells better than the 7G3antibodies. The inhibition is IL-3 dependent, as these antibodies had noinhibitory effect when the cells were grown in the presence of GM-CSF(FIG. 8B).

FIG. 9A shows expression constructs of IL-3Rα (CD123) extra-cellulardomain and chimeric receptor proteins comprising IL-3Rα (gray) and GMRαdomains (white). FIG. 9B shows that the CD123-6 antibody binds primarilyto the CRM domain of IL-3Rα (residues 101-306). FIG. 9C shows that theCD123-3 antibody binds primarily to the CRM domain of IL-3Rα (residues101-306). FIG. 9D shows that the CD123-14 antibody binds exclusively tothe N-terminal domain of IL-3Rα (residues 1-100). FIG. 9E shows that the7G3 antibody binds exclusively to the N-terminal domain of IL-3Rα(residues 1-100). FIG. 9F shows that the 6H6 antibody binds exclusivelyto the N-terminal domain of IL-3Rα (residues 1-100). FIG. 9G shows thatthe 9F5 antibody binds exclusively to the N-terminal domain of IL-3Rα(residues 1-100).

FIG. 10 demonstrates that maytansinoid DM1 conjugate of the resurfacedhuCD123-6Rv1.1 antibody, huCD123-6Rv1.1-CX1-1-DM1, exhibitsdose-dependent cytotoxicity on the growth factor-independentCD123-expressing AML cell line OCI-AML4. The cytotoxicity isCD123-dependent, as evidenced by the ability of excess unconjugatedhuCD123-6 antibody (500 nM) to block the cytotoxicity.

FIG. 11A shows in vitro cytotoxicity of the various resurfacedlysine-linked huCD123-6Rv1.1-IGN conjugates on multiple CD123-positivemalignant cell lines of different origin.

FIG. 11B shows in vitro cytotoxicity of the various lysine- orcysteine-linked huCD123-6-IGN conjugates on multiple CD123-positiveB-ALL cell lines. The non-binding KTI antibody based conjugates areincluded as negative controls.

FIG. 11C shows that the various Lys- or Cys-linked IGN compounds arehighly active on P-gp (P-glycoprotein) positive AML cell lines Kasumi-3and MOLM-1. The control curves with open data points are produced in thepresence of excess unconjugated matching huCD123 antibodies.

FIG. 11D shows that nearly all of the various Lys- or Cys-linkedCD123-IGN conjugates of the invention kill 90% of the AML progenitorcells from 9 AML patient samples at nM or sub-nM concentrations.

FIG. 11E shows that the Cys-linked huCD123-6Gv4.7-CysMab-D5 conjugatekills normal blood cells at concentrations that are >100-fold higherthan those needed to kill AML progenitors. In comparison, Mylotarg doesnot exhibit such preferential killing effect.

FIGS. 12A and 12B show in vitro cytotoxicity of the variouslysine-linked huCD123-6Rv1.1-IGN conjugates on primary cells from AMLpatients. The result from a typical CFU assay for one primary patientsample is presented in FIG. 12A. FIG. 12B shows the IC₉₀ values for allAML patient samples treated with the conjugates.

FIGS. 13A-13C show that the IGN conjugate of CysMab of huCD123-6(huCD123-6Gv4.6-CysMab-D5, filled black circle) is at least as active asthe lysine-linked conjugate (huCD123-6Gv4.6-D2, filled black square) ofthe same antibody towards the AML cell line EOL-1 (FIG. 13A), the B-ALLcell line KOPN-8 (FIG. 13B), and the CML cell line MOLM-1 (FIG. 13C).The dotted curves connecting open data points in each figure representactivity of the respective conjugates (i.e., open circle forhuCD123-6Gv4.6-CysMab-D5, and open square for huCD123-6Gv4.6-D2) in thepresence of blocking concentration (500 nM) of the unconjugatedchCD123-6 antibody.

FIGS. 14A-14C show that SeriMab of huCD123-6(huCD123-6Rv1.1S2-SeriMab-D8, filled black circle) is at least as activeas the lysine-linked conjugate (huCD123-6Rv1.1-D2, filled downward blacktriangle) of the same antibody in AML cell lines SHI-1 (FIG. 14A) andHNT-34 (FIG. 14B), as well as the CML cell line MOLM-1 (FIG. 14C). Thedotted curves connecting open data points in each figure representactivity of the respective conjugates (i.e., open circle forhuCD123-6Rv1.1S2-SeriMab-D8, and open downward triangle forhuCD123-6Rv1.1-D2) in the presence of blocking concentration (500 nM) ofthe unconjugated huCD123-6 antibody.

FIG. 15 shows a schematic drawing to show the general steps that can beused to synthesize a Ser-linkage conjugate of the invention.

FIG. 16 shows a schematic drawing to show the general steps that can beused to synthesize a Ser-linkage conjugate of the invention.

FIG. 17 shows a schematic drawing to show the general steps that can beused to synthesize a Ser-linkage conjugate of the invention.

FIG. 18 shows that the Cys-linked huCD123-6Gv4.7-CysMab-D5 conjugate hashigher activity than gemtuzumab ozogamicin (GO) (also known as Mylotarg)in unselected AML patient samples.

FIG. 19 shows that the Cys-linked huCD123-6Gv4.7-CysMab-D5 conjugatekills normal progenitor cells at concentrations that are >100-foldhigher than those needed to kill AML progenitors. In comparison,Mylotarg and huCD123-6G4.7-CysMab-D5′ (ADC of DNA cross-linker D5′) donot exhibit such preferential killing effect.

FIG. 20 shows in vivo efficacy of CD123-IGN conjugates in the MV4-11 AMLsubcutaneous mice model.

FIG. 21 shows that the Cys-linked huCD123-6Gv4.7-CysMab-D5 conjugate ishighly active on various CD123-positive AML cell lines with poorprognostic factors.

FIG. 22 shows in vivo bioluminescence imaging of mice treated withhuCD123-6Gv4.7-CysMab-D5 conjugate as compared to mice treated withvehicle and control on day 26. Treatment with the conjugatesignificantly reduces tumor burden in mice.

FIG. 23 shows treatment with huCD123-6Gv4.7-CysMab-D5 conjugate extendedsurvival in 6/6 mice as compared to mice treated with vehicle andcontrol.

FIG. 24 shows that the incubation of MV4-11 cells withhuCD123-6Gv4.7-CysMab-D5 conjugate leads to DNA damage, arrest inS-phase of the cell cycle, and apoptosis-mediated cell death.

FIG. 25 shows in vivo efficacy of CD123-IGN conjugates in the Molm-13AML disseminated model.

FIG. 26 shows in vivo efficacy of CD123-IGN conjugates in the EOL-1subcutaneous model.

FIG. 27 shows in vivo efficacy of huCD123-CysMab-D5 conjugate at variousdoses in the EOL-1 subcutaneous model.

FIG. 28 shows in vivo efficacy of huCD123-CysMab-D5 conjugate comparedto the corresponding free drug form of the payload (FGN849 or D5), nakedantibody, control, cytarabine, and azacitidine in the EOL-1 subcutaneousmodel.

FIG. 29 shows in vivo efficacy of CD123-IGN conjugates in the MV4-11 AMLdisseminated model.

FIG. 30 shows in vivo efficacy of CD123-IGN conjugates in the MV4-11 AMLsubcutaneous model.

FIG. 31 shows treatment with huCD123-CysMab-D5 conjugate extendedsurvival in mice as compared to mice treated with vehicle and control.

FIG. 32 shows in vivo tolerability of huCD123-CysMab-D5 andhuCD123-SeriMab-sD1 conjugates in mice.

FIG. 33 shows in vivo tolerability of huCD123-lysine linked-D2 conjugatein mice.

DETAILED DESCRIPTION OF THE INVENTION 1. Definitions

To facilitate an understanding of the present invention, a number ofterms and phrases are defined below.

The terms “(human) IL-3Rα,” “Interleukine-3 Receptor alpha,” or “CD123,”as used interchangeably herein, refers to any native (human) IL-3Rα orCD123, unless otherwise indicated. The CD123 protein is an interleukin3-specific subunit of a heterodimeric cytokine receptor (IL-3 Receptor,or IL-3R). The IL-3R is comprised of a ligand specific alpha subunit,and a signal transducing common beta subunit (also known as CD131)shared by the receptors for interleukin 3 (IL3), colony stimulatingfactor 2 (CSF2/GM-CSF), and interleukin 5 (IL5). The binding ofCD123/IL-3Rα to IL3 depends on the beta subunit. The beta subunit isactivated by the ligand binding, and is required for the biologicalactivities of IL3.

All of these above terms for CD123 can refer to either a protein ornucleic acid sequence as indicated herein. The term “CD123/IL-3Rα”encompasses “full-length,” unprocessed CD123/IL-3Rα, as well as any formof CD123/IL-3Rα that results from processing within the cell. The termalso encompasses naturally occurring variants of CD123/IL-3Rα protein ornucleic acid, e.g., splice variants, allelic variants and isoforms. TheCD123/IL-3Rα polypeptides and polynucleotides described herein can beisolated from a variety of sources, such as from human tissue types orfrom another source, or prepared by recombinant or synthetic methods.Examples of CD123/IL-3Rα sequences include, but are not limited to NCBIreference numbers NP_002174 & NM_002183 (protein and nucleic acidsequences for human CD123 variant 1), and NP_001254642 & NM_001267713(protein and nucleic acid sequences for human CD123 variant 2).

The term “antibody” means an immunoglobulin molecule that recognizes andspecifically binds to a target, such as a protein, polypeptide, peptide,carbohydrate, polynucleotide, lipid, or combinations of the foregoingthrough at least one antigen recognition site within the variable regionof the immunoglobulin molecule. As used herein, the term “antibody”encompasses intact polyclonal antibodies, intact monoclonal antibodies,antibody fragments (such as Fab, Fab′, F(ab′)₂, and Fv fragments),single chain Fv (scFv) mutants, multispecific antibodies such asbispecific antibodies, chimeric antibodies, humanized antibodies, humanantibodies, fusion proteins comprising an antigen determination portionof an antibody, and any other modified immunoglobulin moleculecomprising an antigen recognition site so long as the antibodies exhibitthe desired biological activity. An antibody can be of any of the fivemajor classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, orsubclasses (isotypes) thereof (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 andIgA2), based on the identity of their heavy-chain constant domainsreferred to as alpha, delta, epsilon, gamma, and mu, respectively. Thedifferent classes of immunoglobulins have different and well knownsubunit structures and three-dimensional configurations. Antibodies canbe naked or conjugated to other molecules such as toxins, radioisotopes,etc.

In some embodiments, an antibody is a non-naturally occurring antibody.In some embodiments, an antibody is purified from natural components. Insome embodiments, an antibody is recombinantly produced. In someembodiments, an antibody is produced by a hybridoma.

A “blocking” antibody or an “antagonist” antibody is one which inhibitsor reduces biological activity of the antigen it binds, such asCD123/IL-3Rα. In a certain embodiment, blocking antibodies or antagonistantibodies substantially or completely inhibit the biological activityof the antigen. Desirably, the biological activity is reduced by 10%,20%, 30%, 50%, 70%, 80%, 90%, 95%, or even 100%.

The term “anti-CD123 antibody,” “anti-IL-3Rα antibody” or “an antibodythat (specifically) binds to CD123/IL-3Rα” refers to an antibody that iscapable of binding CD123/IL-3Rα with sufficient affinity such that theantibody is useful as a diagnostic and/or therapeutic agent in targetingCD123/IL-3Rα. Unless otherwise specified, the extent of binding of ananti-CD123/IL-3Rα antibody to an unrelated, non-CD123/IL-3Rα protein isless than about 10% of the binding of the antibody to CD123/IL-3Rα asmeasured, e.g., by a radioimmunoassay (RIA). In certain embodiments, anantibody that binds to CD123/IL-3Rα has a dissociation constant (K_(d))of ≤0.5 nM, ≤0.3 nM, ≤0.1 nM, ≤0.05 nM, or ≤0.01 nM. In one embodiment,the anti-CD123/IL-3Rα antibody does not bind the common beta chainCD131. In one embodiment, the anti-CD123/IL-3Rα antibody does not bindto the same epitope of CD123 that is bound by the known and commerciallyavailable CD123 antibodies such as 7G3 (mouse IgG_(2a)), 6H6 (mouseIgG₁), and 9F5 (mouse IgG₁) (Sun et al., Blood 87(1): 83-92, 1996).

The sequences of anti-CD123/IL-3Rα antibodies and antigen-bindingfragments thereof of the invention are provided in Tables 1-6 below. Thenomenclature for the various antibodies and immuno-conjugates of theinvention are provided separately below.

The term “antibody fragment” refers to a portion of an intact antibodyand refers to the antigenic determining variable regions of an intactantibody. Examples of antibody fragments include, but are not limitedto, Fab, Fab′, F(ab′)₂, and F_(v) fragments, linear antibodies, singlechain antibodies, and multispecific antibodies formed from antibodyfragments. The term “antigen-binding fragment” of an antibody includesone or more fragments of an antibody that retain the ability tospecifically bind to an antigen. It has been shown that theantigen-binding function of an antibody can be performed by certainfragments of a full-length antibody. Examples of binding fragmentsencompassed within the term “antigen-binding fragment” of an antibodyinclude (without limitation): (i) an Fab fragment, a monovalent fragmentconsisting of the V_(L), V_(H), C_(L), and C_(H1) domains (e.g., anantibody digested by papain yields three fragments: two antigen-bindingFab fragments, and one Fc fragment that does not bind antigen); (ii) aF(ab′)₂ fragment, a bivalent fragment comprising two Fab fragmentslinked by a disulfide bridge at the hinge region (e.g., an antibodydigested by pepsin yields two fragments: a bivalent antigen-bindingF(ab′)₂ fragment, and a pFc′ fragment that does not bind antigen) andits related F(ab′) monovalent unit; (iii) a F_(d) fragment consisting ofthe V_(H) and C_(H1) domains (i.e., that portion of the heavy chainwhich is included in the Fab); (iv) a F_(v) fragment consisting of theV_(L) and V_(H) domains of a single arm of an antibody, and the relateddisulfide linked F_(v); (v) a dAb (domain antibody) or sdAb (singledomain antibody) fragment (Ward et al., Nature 341:544-546, 1989), whichconsists of a V_(H) domain; and (vi) an isolated complementaritydetermining region (CDR).

A “monoclonal antibody” refers to a homogeneous antibody populationinvolved in the highly specific recognition and binding of a singleantigenic determinant, or epitope. This is in contrast to polyclonalantibodies that typically include different antibodies directed againstdifferent antigenic determinants. The term “monoclonal antibody”encompasses both intact and full-length monoclonal antibodies as well asantibody fragments (such as Fab, Fab′, F(ab′)₂, F_(v)), single chain(scFv) mutants, fusion proteins comprising an antibody portion, and anyother modified immunoglobulin molecule comprising an antigen recognitionsite. Furthermore, “monoclonal antibody” refers to such antibodies madein any number of manners including but not limited to by hybridoma,phage selection, recombinant expression, and transgenic animals.

The term “humanized antibody” refers to forms of non-human (e.g.,murine) antibodies that are specific immunoglobulin chains, chimericimmunoglobulins, or fragments thereof that contain minimal non-human(e.g., murine) sequences. Typically, humanized antibodies are humanimmunoglobulins in which residues from the complementary determiningregion (CDR) are replaced by residues from the CDR of a non-humanspecies (e.g., mouse, rat, rabbit, hamster) that have the desiredspecificity, affinity, and capability (Jones et al., Nature 321:522-525,1986; Riechmann et al., Nature 332:323-327, 1988; Verhoeyen et al.,Science 239:1534-1536, 1988).

In some instances, the F_(v) framework region (FR) residues of a humanimmunoglobulin are replaced with the corresponding residues in anantibody from a non-human species that has the desired specificity,affinity, and capability. The humanized antibody can be further modifiedby the substitution of additional residues either in the F_(v) frameworkregion and/or within the replaced non-human residues to refine andoptimize antibody specificity, affinity, and/or capability. In general,the humanized antibody will comprise substantially all of at least one,and typically two or three, variable domains containing all orsubstantially all of the CDR regions that correspond to the non-humanimmunoglobulin whereas all or substantially all of the FR regions arethose of a human immunoglobulin consensus sequence. The humanizedantibody can also comprise at least a portion of an immunoglobulinconstant region or domain (F_(c)), typically that of a humanimmunoglobulin. Examples of methods used to generate humanizedantibodies are described in U.S. Pat. Nos. 5,225,539 and 5,639,641,Roguska et al., Proc. Natl. Acad. Sci. USA 91(3):969-973, 1994; andRoguska et al., Protein Eng. 9(10):895-904, 1996 (all incorporatedherein by reference). In some embodiments, a “humanized antibody” is aresurfaced antibody. In some embodiments, a “humanized antibody” is aCDR-grafted antibody.

A “variable region” of an antibody refers to the variable region of theantibody light chain or the variable region of the antibody heavy chain,either alone or in combination. The variable regions of the heavy andlight chain each consist of four framework regions (FR) connected bythree complementarity determining regions (CDRs) also known ashypervariable regions. The CDRs in each chain are held together in closeproximity by the FRs and, with the CDRs from the other chain, contributeto the formation of the antigen-binding site of antibodies. There are atleast two techniques for determining CDRs: (1) an approach based oncross-species sequence variability (i.e., Kabat et al. Sequences ofProteins of Immunological Interest, 5th ed., 1991, National Institutesof Health, Bethesda Md.); and (2) an approach based on crystallographicstudies of antigen-antibody complexes (Al-lazikani et al., J. Molec.Biol. 273:927-948, 1997). In addition, combinations of these twoapproaches are sometimes used in the art to determine CDRs.

The Kabat numbering system is generally used when referring to a residuein the variable domain (approximately residues 1-107 of the light chainand residues 1-113 of the heavy chain) (e.g., Kabat et al., Sequences ofImmunological Interest, 5th Ed., Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991)).

The amino acid position numbering as in Kabat, refers to the numberingsystem used for heavy chain variable domains or light chain variabledomains of the compilation of antibodies in Kabat et al., Sequences ofProteins of Immunological Interest, 5th Ed., Public Health Service,National Institutes of Health, Bethesda, Md. (1991) (incorporated hereinby reference). Using this numbering system, the actual linear amino acidsequence can contain fewer or additional amino acids corresponding to ashortening of, or insertion into, a FR or CDR of the variable domain.For example, a heavy chain variable domain can include a single aminoacid insert (residue 52a according to Kabat) after residue 52 of H2 andinserted residues (e.g., residues 82a, 82b, and 82c, etc. according toKabat) after heavy chain FR residue 82. The Kabat numbering of residuescan be determined for a given antibody by alignment at regions ofhomology of the sequence of the antibody with a “standard” Kabatnumbered sequence. Chothia refers instead to the location of thestructural loops (Chothia and Lesk, J. Mol. Biol. 196:901-917, 1987).The end of the Chothia CDR-H1 loop when numbered using the Kabatnumbering convention varies between H32 and H34 depending on the lengthof the loop. This is because the Kabat numbering scheme places theinsertions at H35A and H35B—if neither 35A nor 35B is present, the loopends at 32; if only 35A is present, the loop ends at 33; if both 35A and35B are present, the loop ends at 34. The AbM hypervariable regionsrepresent a compromise between the Kabat CDRs and Chothia structuralloops, and are used by Oxford Molecular's AbM antibody modelingsoftware.

Loop Kabat AbM Chiothia L1 L24-L34 L24-L34 L24-L34 L2 L50-L56 L50-L56L50-L56 L3 L89-L97 L89-L97 L89-L97 H1 H31-H35B H26-H35B H26-H32 . . . 34(Kabat Numbering) H1 H31-H35 H26-H35 H26-H32 (Chothia Numbering) H2H50-H65 H50-H58 H52-H56 H3 H9S-H102 H95-H102 H95-H102

The term “human antibody” means an antibody produced by a human or anantibody having an amino acid sequence corresponding to an antibodyproduced by a human made using any technique known in the art. Incertain embodiments, the human antibody does not have non-humansequence. This definition of a human antibody includes intact orfull-length antibodies, or antigen-binding fragments thereof.

The term “chimeric antibodies” refers to antibodies wherein the aminoacid sequence of the immunoglobulin molecule is derived from two or morespecies. Typically, the variable region of both light and heavy chainscorresponds to the variable region of antibodies derived from onespecies of mammals (e.g., mouse, rat, rabbit, etc.) with the desiredspecificity, affinity, and capability while the constant regions arehomologous to the sequences in antibodies derived from another (usuallyhuman) to avoid or reduce the chance of eliciting an immune response inthat species (e.g., human). In certain embodiments, chimeric antibodymay include an antibody or antigen-binding fragment thereof comprisingat least one human heavy and/or light chain polypeptide, such as, forexample, an antibody comprising murine light chain and human heavy chainpolypeptides.

The terms “epitope” or “antigenic determinant” are used interchangeablyherein and refer to that portion of an antigen capable of beingrecognized and specifically bound by a particular antibody. When theantigen is a polypeptide, epitopes can be formed both from contiguousamino acids and noncontiguous amino acids juxtaposed by tertiary foldingof a protein. Epitopes formed from contiguous amino acids are typicallyretained upon protein denaturing, whereas epitopes formed by tertiaryfolding are typically lost upon protein denaturing. An epitope typicallyincludes at least 3, and more usually, at least 5 or 8-10 amino acids ina unique spatial conformation.

“Binding affinity” generally refers to the strength of the sum total ofnoncovalent interactions between a single binding site of a molecule(e.g., an antibody) and its binding partner (e.g., an antigen). Unlessindicated otherwise, as used herein, “binding affinity” refers tointrinsic binding affinity which reflects a 1:1 interaction betweenmembers of a binding pair (e.g., antibody and antigen). The affinity ofa molecule X for its partner Y can generally be represented by thedissociation constant (K_(d)) or the half-maximal effectiveconcentration (EC₅₀). Affinity can be measured by common methods knownin the art, including those described herein. Low-affinity antibodiesgenerally bind antigen slowly and tend to dissociate readily, whereashigh-affinity antibodies generally bind antigen faster and tend toremain bound longer. A variety of methods of measuring binding affinityare known in the art, any of which can be used for purposes of thepresent invention. Specific illustrative embodiments are describedherein.

“Or better” when used herein to refer to binding affinity refers to astronger binding between a molecule and its binding partner. “Or better”when used herein refers to a stronger binding, represented by a smallernumerical K_(d) value. For example, an antibody which has an affinityfor an antigen of “0.3 nM or better,” the antibody's affinity for theantigen is ≤0.3 nM, e.g., 0.29 nM, 0.28 nM, 0.27 nM etc., or any valueequal to or less than 0.3 nM. In one embodiment, the antibody's affinityas determined by a K_(d) will be between about 10⁻³ to about 10⁻¹² M,between about 10⁻⁶ to about 10⁻¹¹ M, between about 10⁻⁶ to about 10⁻¹⁰M, between about 10⁻⁶ to about 10⁻⁹ M, between about 10⁻⁶ to about 10⁻⁸M, or between about 10⁻⁶ to about 10⁻⁷ M.

By “specifically binds,” it is generally meant that an antibody binds toan epitope via its antigen-binding domain, and that the binding entailssome complementarity between the antigen-binding domain and the epitope.According to this definition, an antibody is said to “specifically bind”to an epitope when it binds to that epitope, via its antigen-bindingdomain more readily than it would bind to a random, unrelated epitope.The term “specificity” is used herein to qualify the relative affinityby which a certain antibody binds to a certain epitope. For example,antibody “A” may be deemed to have a higher specificity for a givenepitope than antibody “B,” or antibody “A” may be said to bind toepitope “C” with a higher specificity than it has for related epitope“D.”

In certain embodiments, an antibody or antigen-binding fragment of theinvention “specifically binds” to a CD123 antigen, in that it has ahigher binding specificity to the CD123 antigen (from any species) thanthat to a non-CD123 antigen. In certain embodiments, an antibody orantigen-binding fragment of the invention “specifically binds” to ahuman CD123 antigen, in that it has a higher binding specificity to thehuman CD123 antigen than that to a non-human CD123 antigen (e.g., amouse or a rat CD123).

By “preferentially binds,” it is meant that the antibody specificallybinds to an epitope more readily than it would bind to a related,similar, homologous, or analogous epitope. Thus, an antibody which“preferentially binds” to a given epitope would more likely bind to thatepitope than to a related epitope, even though such an antibody maycross-react with the related epitope. For example, in certainembodiments, an antibody or antigen-binding fragment of the invention“preferentially binds” to a human CD123 antigen over a mouse CD123.

An antibody is said to “competitively inhibit” binding of a referenceantibody to a given epitope if it preferentially binds to that epitopeto the extent that it blocks, to some degree, binding of the referenceantibody to the epitope. Competitive inhibition may be determined by anymethod known in the art, for example, competition ELISA assays. Anantibody may be said to competitively inhibit binding of the referenceantibody to a given epitope by at least 90%, at least 80%, at least 70%,at least 60%, or at least 50%.

The phrase “substantially similar,” or “substantially the same,” as usedherein, denotes a sufficiently high degree of similarity between twonumeric values (generally one associated with an antibody of theinvention and the other associated with a reference/comparator antibody)such that one of skill in the art would consider the difference betweenthe two values to be of little or no biological and/or statisticalsignificance within the context of the biological characteristicsmeasured by said values (e.g., K_(d) values). The difference betweensaid two values is less than about 50%, less than about 40%, less thanabout 30%, less than about 20%, or less than about 10% as a function ofthe value for the reference/comparator antibody.

A polypeptide, antibody, polynucleotide, vector, cell, or compositionwhich is “isolated” is a polypeptide, antibody, polynucleotide, vector,cell, or composition which is in a form not found in nature. Isolatedpolypeptides, antibodies, polynucleotides, vectors, cells orcompositions include those which have been purified to a degree thatthey are no longer in a form in which they are found in nature. In someembodiments, an antibody, polynucleotide, vector, cell, or compositionwhich is isolated is substantially pure.

As used herein, “substantially pure” refers to material which is atleast 50% pure (i.e., free from contaminants), at least 90% pure, atleast 95% pure, at least 98% pure, or at least 99% pure.

The term “immunoconjugate,” “conjugate,” or “ADC” as used herein refersto a compound or a derivative thereof that is linked to a cell bindingagent (i.e., an anti-CD123/IL-3Rα antibody or fragment thereof) and isdefined by a generic formula: A-L-C, wherein C=cytotoxin, L=linker, andA=cell binding agent (CBA), such as anti-CD123/IL-3Rα antibody orantibody fragment. Immunoconjugates can also be defined by the genericformula in reverse order: C-L-A.

A “linker” is any chemical moiety that is capable of linking a compound,usually a drug, such as a cytotoxic agent described herein (e.g.,maytansinoid or IGN (indolinobenzodiazepine) compounds), to acell-binding agent such as an anti-CD123/IL-3Rα antibody or a fragmentthereof in a stable, covalent manner. Linkers can be susceptible to orbe substantially resistant to acid-induced cleavage, light-inducedcleavage, peptidase-induced cleavage, esterase-induced cleavage, anddisulfide bond cleavage, at conditions under which the compound or theantibody remains active. Suitable linkers are well known in the art andinclude, for example, disulfide groups, thioether groups, acid labilegroups, photolabile groups, peptidase labile groups and esterase labilegroups. Linkers also include charged linkers, and hydrophilic formsthereof as described herein and know in the art.

The terms “elevated” CD123/IL-3Rα, “increased expression” ofCD123/IL-3Rα and “overexpression” of CD123/IL-3Rα refer to a samplewhich contains elevated levels of CD123 expression. The CD123 can beelevated, increased, or overexpressed as compared to a control value(e.g., expression level in a biological sample, tissue, or cell from asubject without cancer, a sample or cancer known to express no or lowCD123/IL-3Rα, a normal sample, or a cancer that does not have elevatedCD123/IL-3Rα values). For example, a sample (e.g., one from ahematological cancer such as leukemia and lymphoma) with increasedexpression can contain an increase of at least 2-, 3-, 4-, 5-, 10-, 15-,20-, 25-, 30-, or at least 50-fold relative to a control/normal values.

A “reference sample” can be used to correlate and compare the resultsobtained in the methods of the invention from a test sample. Referencesamples can be cells (e.g., cell lines, cell pellets) or tissue. TheCD123/IL-3Rα levels in the “reference sample” can be an absolute orrelative amount, a range of amount, a minimum and/or maximum amount, amean amount, and/or a median amount of CD123/IL-3Rα. A “referencesample” can also serve as a baseline of CD123/IL-3Rα expression to whichthe test sample is compared. The “reference sample” can include a priorsample or baseline sample from the same patient, a normal reference witha known level of CD123/IL-3Rα expression, or a reference from a relevantpatient population with a known level of CD123/IL-3Rα expression.CD123/IL-3Rα levels can also be expressed as values in a standard curve.A standard curve is a quantitative method of plotting assay data todetermine the concentration of CD123/IL-3Rα in a sample. In oneembodiment, a reference sample is an antigen standard comprisingpurified CD123/IL-3Rα. The diagnostic methods of the invention caninvolve a comparison between expression levels of CD123/IL-3Rα in a testsample and a “reference value.” In some embodiments, the reference valueis the expression level of the CD123/IL-3Rα in a reference sample. Areference value can be a predetermined value and can also be determinedfrom reference samples (e.g., control biological samples or referencesamples) tested in parallel with the test samples. A reference value canbe a single cut-off value, such as a median or mean or a range ofvalues, such as a confidence interval. Reference values can beestablished for various subgroups of individuals.

The term “primary antibody” herein refers to an antibody that bindsspecifically to the target protein antigen in a sample. A primaryantibody is generally the first antibody used in an ELISA assay or IHCprocedure. In one embodiment, the primary antibody is the only antibodyused in an IHC procedure.

The term “secondary antibody” herein refers to an antibody that bindsspecifically to a primary antibody, thereby forming a bridge or linkbetween the primary antibody and a subsequent reagent, if any. Thesecondary antibody is generally the second antibody used in animmunohistochemical procedure.

A “sample” or “biological sample” of the present invention is ofbiological origin, in specific embodiments, such as from eukaryoticorganisms. In some embodiments, the sample is a human sample, but animalsamples may also be used. Non-limiting sources of a sample for use inthe present invention include solid tissue, biopsy aspirates, ascites,fluidic extracts, blood, plasma, serum, spinal fluid, lymph fluid, theexternal sections of the skin, respiratory, intestinal, andgenitourinary tracts, tears, saliva, milk, tumors, organs, cell culturesand/or cell culture constituents, for example. A “cancerous sample” is asample that contains a cancerous cell. The method can be used to examinean aspect of expression of CD123/IL-3Rα or a state of a sample,including, but not limited to, comparing different types of cells ortissues, comparing different developmental stages, and detecting ordetermining the presence and/or type of disease or abnormality.

As used herein, the term “capture reagent” refers to a reagent capableof binding and capturing a target molecule in a sample such that undersuitable condition, the capture reagent-target molecule complex can beseparated from the rest of the sample. In one embodiment, the capturereagent is immobilized. In one embodiment, the capture reagent in asandwich immunoassay is an antibody or a mixture of different antibodiesagainst a target antigen.

As used herein, the term “detectable antibody” refers to an antibodythat is capable of being detected either directly through a labelamplified by a detection means, or indirectly through, e.g., anotherantibody that is labeled. For direct labeling, the antibody is typicallyconjugated to a moiety that is detectable by some means. In oneembodiment, the detectable antibody is a biotinylated antibody.

As used herein, the term “detection means” refers to a moiety ortechnique used to detect the presence of the detectable antibody andincludes detection agents that amplify the immobilized label such aslabel captured onto a microtiter plate. In one embodiment, the detectionmeans is a fluorimetric detection agent such as avidin or streptavidin.

Commonly a “sandwich ELISA” employs the following steps: (1) microtiterplate is coated with a capture antibody; (2) sample is added, and anyantigen present binds to capture antibody; (3) detecting antibody isadded and binds to antigen; (4) enzyme-linked secondary antibody isadded and binds to detecting antibody; and (5) substrate is added and isconverted by enzyme to detectable form.

The word “label” when used herein refers to a detectable compound orcomposition which is conjugated directly or indirectly to the antibodyso as to generate a “labeled” antibody. The label can be detectable byitself (e.g. radioisotope labels or fluorescent labels) or, in the caseof an enzymatic label, can catalyze chemical alteration of a substratecompound or composition which is detectable.

By “correlate” or “correlating” is meant comparing, in any way, theperformance and/or results of a first analysis with the performanceand/or results of a second analysis. For example, one may use theresults of a first analysis in carrying out the second analysis and/orone may use the results of a first analysis to determine whether asecond analysis should be performed and/or one may compare the resultsof a first analysis with the results of a second analysis. In oneembodiment, increased expression of CD123/IL-3Rα correlates withincreased likelihood of effectiveness of a CD123/IL-3Rα-targetingtherapy.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals in which a population of cells arecharacterized by unregulated cell growth. “Tumor” and “neoplasm” referto one or more cells that result from excessive cell growth orproliferation, either benign (noncancerous) or malignant (cancerous)including pre-cancerous lesions.

Examples of cancer include lymphoma and leukemia. Examples of cancer ortumorigenic diseases which can be treated and/or prevented by themethods and reagents (e.g., anti-CD123 antibody, antigen-bindingfragment thereof, or immuno-conjugate thereof) of the invention includeAML, CML, ALL (e.g., B-ALL), CLL, myelodysplastic syndrome, basicplasmacytoid DC neoplasm (BPDCN) leukemia, B-cell lymphomas includingnon-Hodgkin lymphomas (NHL), precursor B-cell lymphoblasticleukemia/lymphoma and mature B-cell neoplasms, such as B-cell chroniclymphocytic leukemia (B-CLL)/small lymphocytic lymphoma (SLL), B-cellprolymphocytic leukemia, lymphoplasmacytic lymphoma, mantle celllymphoma (MCL), follicular lymphoma (FL), including low-grade,intermediate-grade and high-grade FL, cutaneous follicle centerlymphoma, marginal zone B-cell lymphoma (MALT type, nodal and splenictype), hairy cell leukemia (HCL), diffuse large B-cell lymphoma,Burkitt's lymphoma, plasmacytoma, plasma cell myeloma, post-transplantlymphoproliferative disorder, Waldenstrom's macroglobulinemia,anaplastic large-cell lymphoma (ALCL), and Hodgkin's leukemia (HL).

Cancers also encompass cancers which contain cells having elevatedCD123/IL-3Rα expression levels. Such CD123/IL-3Rα-elevated cancersinclude, but are not limited to, AML, CML, ALL (e.g., B-ALL), and CLL.

The terms “cancer cell,” “tumor cell,” and grammatical equivalents referto the total population of cells derived from a tumor or a pre-cancerouslesion, including both non-tumorigenic cells, which comprise the bulk ofthe tumor cell population, and tumorigenic stem cells (cancer stemcells). As used herein, the term “tumor cell” will be modified by theterm “non-tumorigenic” when referring solely to those tumor cellslacking the capacity to renew and differentiate to distinguish thosetumor cells from cancer stem cells.

The term “subject” refers to any animal (e.g., a mammal), including, butnot limited to humans, non-human primates, rodents, and the like, whichis to be the recipient of a particular treatment. Typically, the terms“subject” and “patient” are used interchangeably herein in reference toa human subject.

Administration “in combination with” one or more further therapeuticagents includes simultaneous (concurrent) and consecutive administrationin any order.

The term “pharmaceutical formulation” refers to a preparation which isin such form as to permit the biological activity of the activeingredient to be effective, and which contains no additional componentswhich are unacceptably toxic to a subject to which the formulation wouldbe administered. Such formulation can be sterile.

An “effective amount” of an antibody or immunoconjugate as disclosedherein is an amount sufficient to carry out a specifically statedpurpose. An “effective amount” can be determined empirically and in aroutine manner, in relation to the stated purpose.

The term “therapeutically effective amount” refers to an amount of anantibody or other drug effective to “treat” a disease or disorder in asubject or mammal. In the case of cancer, the therapeutically effectiveamount of the drug can reduce the number of cancer cells; reduce thetumor size; inhibit (i.e., slow to some extent and in a certainembodiment, stop) cancer cell infiltration into peripheral organs;inhibit (i.e., slow to some extent and in a certain embodiment, stop)tumor metastasis; inhibit, to some extent, tumor growth; relieve to someextent one or more of the symptoms associated with the cancer; and/orresult in a favorable response such as increased progression-freesurvival (PFS), disease-free survival (DFS), or overall survival (OS),complete response (CR), partial response (PR), or, in some cases, stabledisease (SD), a decrease in progressive disease (PD), a reduced time toprogression (TTP), or any combination thereof. See the definition hereinof “treating.” To the extent the drug can prevent growth and/or killexisting cancer cells, it can be cytostatic and/or cytotoxic.

A “prophylactically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredprophylactic result. Typically but not necessarily, since a prophylacticdose is used in subjects prior to or at an earlier stage of disease, theprophylactically effective amount will be less than the therapeuticallyeffective amount.

The term “respond favorably” generally refers to causing a beneficialstate in a subject. With respect to cancer treatment, the term refers toproviding a therapeutic effect on the subject. Positive therapeuticeffects in cancer can be measured in a number of ways (See, W. A. Weber,J. Nucl. Med. 50: 1S-10S (2009)). For example, tumor growth inhibition,molecular marker expression, serum marker expression, and molecularimaging techniques can all be used to assess therapeutic efficacy of ananti-cancer therapeutic. With respect to tumor growth inhibition,according to NCI standards, a T/C<42% is the minimum level of anti-tumoractivity. A T/C<10% is considered a high anti-tumor activity level, withT/C (%)=Median tumor volume of the treated/Median tumor volume of thecontrol×100. A favorable response can be assessed, for example, byincreased progression-free survival (PFS), disease-free survival (DFS),or overall survival (OS), complete response (CR), partial response (PR),or, in some cases, stable disease (SD), a decrease in progressivedisease (PD), a reduced time to progression (TTP), or any combinationthereof.

PFS, DFS, and OS can be measured by standards set by the National CancerInstitute and the U.S. Food and Drug Administration for the approval ofnew drugs. See Johnson et al., (2003) J. Clin. Oncol. 21(7): 1404-1411.

“Progression free survival” (PFS) refers to the time from enrollment todisease progression or death. PFS is generally measured using theKaplan-Meier method and Response Evaluation Criteria in Solid Tumors(RECIST) 1.1 standards. Generally, progression free survival refers tothe situation wherein a patient remains alive, without the cancergetting worse.

A “complete response” or “complete remission” or “CR” indicates thedisappearance of all signs of tumor or cancer in response to treatment.This does not always mean the cancer has been cured.

A “partial response” or “PR” refers to a decrease in the size or volumeof one or more tumors or lesions, or in the extent of cancer in thebody, in response to treatment.

“Stable disease” refers to disease without progression or relapse. Instable disease there is neither sufficient tumor shrinkage to qualifyfor partial response nor sufficient tumor increase to qualify asprogressive disease.

“Progressive disease” refers to the appearance of one more new lesionsor tumors and/or the unequivocal progression of existing non-targetlesions. Progressive disease can also refer to a tumor growth of morethan 20 percent since treatment began, either due to an increases inmass or in spread of the tumor.

“Disease free survival” (DFS) refers to the length of time during andafter treatment that the patient remains free of disease.

“Overall Survival” (OS) refers to the time from patient enrollment todeath or censored at the date last known alive. OS includes aprolongation in life expectancy as compared to naive or untreatedindividuals or patients. Overall survival refers to the situationwherein a patient remains alive for a defined period of time, such asone year, five years, etc., e.g., from the time of diagnosis ortreatment.

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer, regardless of mechanism of action. Terms such as“treating” or “treatment” or “to treat” or “alleviating” or “toalleviate” refer to therapeutic measures that cure, slow down, lessensymptoms of, and/or halt progression of a diagnosed pathologic conditionor disorder. Thus, those in need of treatment include those alreadydiagnosed with the disorder, and may also include those who have minimalresidual disease, or resistant disease, or relapsed disease. In certainembodiments, a subject is successfully “treated” for cancer according tothe methods of the present invention if the patient shows one or more ofthe following: a reduction in the number of or complete absence ofcancer cells; a reduction in the tumor size; inhibition of or an absenceof cancer cell infiltration into peripheral organs including, forexample, the spread of cancer into soft tissue and bone; inhibition ofor an absence of tumor metastasis; inhibition or an absence of tumorgrowth; relief of one or more symptoms associated with the specificcancer; reduced morbidity and mortality; improvement in quality of life;reduction in tumorigenicity, tumorigenic frequency, or tumorigeniccapacity, of a tumor; reduction in the number or frequency of cancerstem cells in a tumor; differentiation of tumorigenic cells to anon-tumorigenic state; increased progression-free survival (PFS),disease-free survival (DFS), or overall survival (OS), complete response(CR), partial response (PR), stable disease (SD), a decrease inprogressive disease (PD), a reduced time to progression (TTP), or anycombination thereof.

Prophylactic or preventative measures refer to measures that preventand/or slow the development of a targeted pathologic condition ordisorder. Thus, those in need of prophylactic or preventative measuresinclude those prone to have the disorder and those in whom the disorderis to be prevented.

Prophylactic or preventative measures refer to therapeutic measures thatprevent and/or slow the development of a targeted pathologic conditionor disorder. Thus, those in need of prophylactic or preventativemeasures include those prone to have the disorder and those in whom thedisorder is to be prevented.

As used herein, the term “healthcare provider” refers to individuals orinstitutions which directly interact with and administer to livingsubjects, e.g., human patients. Non-limiting examples of healthcareproviders include doctors, nurses, technicians, therapist, pharmacists,counselors, alternative medicine practitioners, medical facilities,doctor's offices, hospitals, emergency rooms, clinics, urgent carecenters, alternative medicine clinics/facilities, and any other entityproviding general and/or specialized treatment, assessment, maintenance,therapy, medication, and/or advice relating to all, or any portion of, apatient's state of health, including but not limited to general medical,specialized medical, surgical, and/or any other type of treatment,assessment, maintenance, therapy, medication and/or advice.

In some aspects, a healthcare provider can administer or instructanother healthcare provider to administer a therapy to treat a cancer.“Administration” of a therapy, as used herein, includes prescribing atherapy to a subject as well as delivering, applying, or giving thetherapy to a subject. A healthcare provider can implement or instructanother healthcare provider or patient to perform the following actions:obtain a sample, process a sample, submit a sample, receive a sample,transfer a sample, analyze or measure a sample, quantify a sample,provide the results obtained after analyzing/measuring/quantifying asample, receive the results obtained afteranalyzing/measuring/quantifying a sample, compare/score the resultsobtained after analyzing/measuring/quantifying one or more samples,provide the comparison/score from one or more samples, obtain thecomparison/score from one or more samples, administer a therapy ortherapeutic agent (e.g., a CD123/IL-3Rα binding agent), commence theadministration of a therapy, cease the administration of a therapy,continue the administration of a therapy, temporarily interrupt theadministration of a therapy, increase the amount of an administeredtherapeutic agent, decrease the amount of an administered therapeuticagent, continue the administration of an amount of a therapeutic agent,increase the frequency of administration of a therapeutic agent,decrease the frequency of administration of a therapeutic agent,maintain the same dosing frequency on a therapeutic agent, replace atherapy or therapeutic agent by at least another therapy or therapeuticagent, combine a therapy or therapeutic agent with at least anothertherapy or additional therapeutic agent. These actions can be performedby a healthcare provider automatically using a computer-implementedmethod (e.g., via a web service or stand-alone computer system).

“Polynucleotide” or “nucleic acid,” as used interchangeably herein,refer to polymers of nucleotides of any length, and include DNA and RNA.The nucleotides can be deoxyribonucleotides, ribonucleotides, modifiednucleotides or bases, and/or their analogs, or any substrate that can beincorporated into a polymer by DNA or RNA polymerase. A polynucleotidecan comprise modified nucleotides, such as methylated nucleotides andtheir analogs. If present, modification to the nucleotide structure canbe imparted before or after assembly of the polymer. The sequence ofnucleotides can be interrupted by non-nucleotide components. Apolynucleotide can be further modified after polymerization, such as byconjugation with a labeling component. Other types of modificationsinclude, for example, “caps,” substitution of one or more of thenaturally occurring nucleotides with an analog, internucleotidemodifications such as, for example, those with uncharged linkages (e.g.,methyl phosphonates, phosphotriesters, phosphoamidates, carbamates,etc.) and with charged linkages (e.g., phosphorothioates,phosphorodithioates, etc.), those containing pendant moieties, such as,for example, proteins (e.g., nucleases, toxins, antibodies, signalpeptides, ply-L-lysine, etc.), those with intercalators (e.g., acridine,psoralen, etc.), those containing chelators (e.g., metals, radioactivemetals, boron, oxidative metals, etc.), those containing alkylators,those with modified linkages (e.g., alpha anomeric nucleic acids, etc.),as well as unmodified forms of the polynucleotide(s). Further, any ofthe hydroxyl groups ordinarily present in the sugars can be replaced,for example, by phosphonate groups, phosphate groups, protected bystandard protecting groups, or activated to prepare additional linkagesto additional nucleotides, or can be conjugated to solid supports. The5′ and 3′ terminal OH can be phosphorylated or substituted with aminesor organic capping group moieties of from 1 to 20 carbon atoms. Otherhydroxyls can also be derivatized to standard protecting groups.Polynucleotides can also contain analogous forms of ribose ordeoxyribose sugars that are generally known in the art, including, forexample, 2′-O-methyl-, 2′-O-allyl, 2′-fluoro- or 2′-azido-ribose,carbocyclic sugar analogs, alpha-anomeric sugars, epimeric sugars suchas arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars,sedoheptuloses, acyclic analogs and abasic nucleoside analogs such asmethyl riboside. One or more phosphodiester linkages can be replaced byalternative linking groups. These alternative linking groups include,but are not limited to, embodiments wherein phosphate is replaced byP(O)S (“thioate”), P(S)S (“dithioate”), (O)NR₂ (“amidate”), P(O)R,P(O)OR*, CO or CH₂ (“formacetal”), in which each R or R is independentlyH or substituted or unsubstituted alkyl (1-20 C) optionally containingan ether (—O—) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl oraraldyl. Not all linkages in a polynucleotide need be identical. Thepreceding description applies to all polynucleotides referred to herein,including RNA and DNA.

The term “vector” means a construct, which is capable of delivering, andexpressing, one or more gene(s) or sequence(s) of interest in a hostcell. Examples of vectors include, but are not limited to, viralvectors, naked DNA or RNA expression vectors, plasmid, cosmid or phagevectors, DNA or RNA expression vectors associated with cationiccondensing agents, DNA or RNA expression vectors encapsulated inliposomes, and certain eukaryotic cells, such as producer cells.

The terms “polypeptide,” “peptide,” and “protein” are usedinterchangeably herein to refer to polymers of amino acids of anylength. The polymer can be linear or branched, it can comprise modifiedamino acids, and it can be interrupted by non-amino acids. The termsalso encompass an amino acid polymer that has been modified naturally orby intervention; for example, disulfide bond formation, glycosylation,lipidation, acetylation, phosphorylation, or any other manipulation ormodification, such as conjugation with a labeling component. Alsoincluded within the definition are, for example, polypeptides containingone or more analogs of an amino acid (including, for example, unnaturalamino acids, etc.), as well as other modifications known in the art. Itis understood that, because the polypeptides of this invention are basedupon antibodies, in certain embodiments, the polypeptides can occur assingle chains or associated chains. In some embodiments, a polypeptide,peptide, or protein is non-naturally occurring. In some embodiments, apolypeptide, peptide, or protein is purified from other naturallyoccurring components. In some embodiments, the polypeptide, peptide, orprotein is recombinantly produced.

The terms “identical” or percent “identity” in the context of two ormore nucleic acids or polypeptides, refer to two or more sequences orsubsequences that are the same or have a specified percentage ofnucleotides or amino acid residues that are the same, when compared andaligned (introducing gaps, if necessary) for maximum correspondence, notconsidering any conservative amino acid substitutions as part of thesequence identity. The percent identity can be measured using sequencecomparison software or algorithms or by visual inspection. Variousalgorithms and software are known in the art that can be used to obtainalignments of amino acid or nucleotide sequences. One such non-limitingexample of a sequence alignment algorithm is the algorithm described inKarlin et al., Proc. Natl. Acad. Sci. 87:2264-2268, 1990, as modified inKarlin et al., Proc. Natl. Acad. Sci. 90:5873-5877, 1993, andincorporated into the NBLAST and XBLAST programs (Altschul et al.,Nucleic Acids Res. 25:3389-3402, 1991). In certain embodiments, GappedBLAST can be used as described in Altschul et al., Nucleic Acids Res.25:3389-3402, 1997; BLAST-2, WU-BLAST-2 (Altschul et al., Methods inEnzymology 266:460-480, 1996), ALIGN, ALIGN-2 (Genentech, South SanFrancisco, Calif.) or Megalign (DNASTAR) are additional publiclyavailable software programs that can be used to align sequences. Incertain embodiments, the percent identity between two nucleotidesequences is determined using the GAP program in GCG software (e.g.,using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 90and a length weight of 1, 2, 3, 4, 5, or 6). In certain alternativeembodiments, the GAP program in the GCG software package, whichincorporates the algorithm of Needleman and Wunsch (J. Mol. Biol.(48):444-453, 1970) can be used to determine the percent identitybetween two amino acid sequences (e.g., using either a Blossum 62 matrixor a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and alength weight of 1, 2, 3, 4, 5). Alternatively, in certain embodiments,the percent identity between nucleotide or amino acid sequences isdetermined using the algorithm of Myers and Miller (CABIOS, 4:11-17,1989). For example, the percent identity can be determined using theALIGN program (version 2.0) and using a PAM120 with residue table, a gaplength penalty of 12 and a gap penalty of 4. Appropriate parameters formaximal alignment by particular alignment software can be determined byone skilled in the art. In certain embodiments, the default parametersof the alignment software are used. In certain embodiments, thepercentage identity “X” of a first amino acid sequence to a secondsequence amino acid is calculated as 100×(Y/Z), where Y is the number ofamino acid residues scored as identical matches in the alignment of thefirst and second sequences (as aligned by visual inspection or aparticular sequence alignment program) and Z is the total number ofresidues in the second sequence. If the length of a first sequence islonger than the second sequence, the percent identity of the firstsequence to the second sequence will be longer than the percent identityof the second sequence to the first sequence.

As a non-limiting example, whether any particular polynucleotide has acertain percentage sequence identity (e.g., is at least 80% identical,at least 85% identical, at least 90% identical, and in some embodiments,at least 95%, 96%, 97%, 98%, or 99% identical) to a reference sequencecan, in certain embodiments, be determined using the Bestfit program(Wisconsin Sequence Analysis Package, Version 8 for Unix, GeneticsComputer Group, University Research Park, 575 Science Drive, Madison,Wis. 53711). Bestfit uses the local homology algorithm of Smith andWaterman, Advances in Applied Mathematics 2: 482-489, 1981, to find thebest segment of homology between two sequences. When using Bestfit orany other sequence alignment program to determine whether a particularsequence is, for instance, 95% identical to a reference sequenceaccording to the present invention, the parameters are set such that thepercentage of identity is calculated over the full length of thereference nucleotide sequence and that gaps in homology of up to 5% ofthe total number of nucleotides in the reference sequence are allowed.

In some embodiments, two nucleic acids or polypeptides of the inventionare substantially identical, meaning they have at least 70%, at least75%, at least 80%, at least 85%, at least 90%, and in some embodimentsat least 95%, 96%, 97%, 98%, 99% nucleotide or amino acid residueidentity, when compared and aligned for maximum correspondence, asmeasured using a sequence comparison algorithm or by visual inspection.In certain embodiments, identity exists over a region of the sequencesthat is at least about 10, about 20, about 40-60 residues in length orany integral value therebetween, or over a longer region than 60-80residues, at least about 90-100 residues, or the sequences aresubstantially identical over the full length of the sequences beingcompared, such as the coding region of a nucleotide sequence forexample.

A “conservative amino acid substitution” is one in which one amino acidresidue is replaced with another amino acid residue having a similarside chain. Families of amino acid residues having similar side chainshave been defined in the art, including basic side chains (e.g., lysine,arginine, histidine), acidic side chains (e.g., aspartic acid, glutamicacid), uncharged polar side chains (e.g., asparagine, glutamine, serine,threonine, tyrosine, cysteine), nonpolar side chains (e.g., glycine,alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). For example, substitution of aphenylalanine for a tyrosine is a conservative substitution. In certainembodiments, conservative substitutions in the sequences of thepolypeptides and antibodies of the invention do not abrogate the bindingof the polypeptide or antibody containing the amino acid sequence, tothe antigen(s), i.e., the CD123/IL-3Rα to which the polypeptide orantibody binds. Methods of identifying nucleotide and amino acidconservative substitutions which do not eliminate antigen-binding arewell-known in the art (see, e.g., Brummell et al., Biochem.32:1180-1187, 1993; Kobayashi et al., Protein Eng. 12(10):879-884, 1999;and Burks et al., Proc. Natl. Acad. Sci. USA 94:412-417, 1997).

As used herein, “P-glycoprotein 1,” also known as “permeabilityglycoprotein,” “P-gp or Pgp,” “multidrug resistance protein 1 (MDR1),”“ATP-binding cassette sub-family B member 1 (ABCB1),” or “cluster ofdifferentiation 243 (CD243),” is an ABC-transporter of the MDR/TAPsubfamily that transports a wide variety of substrates across extra- andintracellular membranes. It is an ATP-dependent efflux pump with broadsubstrate specificity. P-gp is extensively distributed and expressed inthe intestinal epithelium where it pumps xenobiotics (such as toxins ordrugs) back into the intestinal lumen, in liver cells where it pumpsthem into bile ducts, in the cells of the proximal tubule of the kidneywhere it pumps them into urine-conducting ducts, and in the capillaryendothelial cells composing the blood-brain barrier and blood-testisbarrier, where it pumps them back into the capillaries. Some cancercells also express large amounts of P-gp, which renders these cancersmulti-drug resistant.

“Alkyl” as used herein refers to a saturated linear or branched-chainmonovalent hydrocarbon radical of one to twenty carbon atoms. Examplesof alkyl include, but are not limited to, methyl, ethyl, 1-propyl,2-propyl, 1-butyl, 2-methyl-1-propyl, —CH₂CH(CH₃)₂), 2-butyl,2-methyl-2-propyl, 1-pentyl, 2-pentyl 3-pentyl, 2-methyl-2-butyl,3-methyl-2-butyl, 3-methyl-1-butyl, 2-methyl-1-butyl, 1-hexyl), 2-hexyl,3-hexyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl,3-methyl-3-pentyl, 2-methyl-3-pentyl, 2,3-dimethyl-2-butyl,3,3-dimethyl-2-butyl, 1-heptyl, 1-octyl, and the like. Preferably, thealkyl has one to ten carbon atoms. More preferably, the alkyl has one tofour carbon atoms.

The number of carbon atoms in a group can be specified herein by theprefix “C_(x-xx)”, wherein x and xx are integers. For example,“C₁₋₄alkyl” is an alkyl group having from 1 to 4 carbon atoms.

The term “compound” or “cytotoxic compound,” are used interchangeably.They are intended to include compounds for which a structure or formulaor any derivative thereof has been disclosed in the present invention ora structure or formula or any derivative thereof that has beenincorporated by reference. The term also includes, stereoisomers,geometric isomers, tautomers, solvates, metabolites, and salts (e.g.,pharmaceutically acceptable salts) of a compound of all the formulaedisclosed in the present invention. The term also includes any solvates,hydrates, and polymorphs of any of the foregoing. The specificrecitation of “stereoisomers,” “geometric isomers,” “tautomers,”“solvates,” “metabolites,” “salt”, “conjugates,” “conjugates salt,”“solvate,” “hydrate,” or “polymorph” in certain aspects of the inventiondescribed in this application shall not be interpreted as an intendedomission of these forms in other aspects of the invention where the term“compound” is used without recitation of these other forms.

The term “chiral” refers to molecules that have the property ofnon-superimposability of the mirror image partner, while the term“achiral” refers to molecules that are superimposable on their mirrorimage partner.

The term “stereoisomer” refers to compounds that have identical chemicalconstitution and connectivity, but different orientations of their atomsin space that cannot be interconverted by rotation about single bonds.

“Diastereomer” refers to a stereoisomer with two or more centers ofchirality and whose molecules are not mirror images of one another.Diastereomers have different physical properties, e.g. melting points,boiling points, spectral properties, and reactivities. Mixtures ofdiastereomers can separate under high resolution analytical proceduressuch as crystallization, electrophoresis and chromatography.

“Enantiomers” refer to two stereoisomers of a compound that arenon-superimposable mirror images of one another.

Stereochemical definitions and conventions used herein generally followS. P. Parker, Ed., McGraw-Hill, Dictionary of Chemical Terms (1984)McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S.,Stereochemistry of Organic Compounds, John Wiley & Sons, Inc., New York,1994. The compounds of the invention can contain asymmetric or chiralcenters, and therefore exist in different stereoisomeric forms. It isintended that all stereoisomeric forms of the compounds of theinvention, including but not limited to, diastereomers, enantiomers andatropisomers, as well as mixtures thereof such as racemic mixtures, formpart of the present invention. Many organic compounds exist in opticallyactive forms, i.e., they have the ability to rotate the plane ofplane-polarized light. In describing an optically active compound, theprefixes D and L, or R and S, are used to denote the absoluteconfiguration of the molecule about its chiral center(s). The prefixes dand I or (+) and (−) are employed to designate the sign of rotation ofplane-polarized light by the compound, with (−) or 1 meaning that thecompound is levorotatory. A compound prefixed with (+) or d isdextrorotatory. For a given chemical structure, these stereoisomers areidentical except that they are mirror images of one another. A specificstereoisomer can also be referred to as an enantiomer, and a mixture ofsuch isomers is often called an enantiomeric mixture. A 50:50 mixture ofenantiomers is referred to as a racemic mixture or a racemate, which canoccur where there has been no stereoselection or stereospecificity in achemical reaction or process. The terms “racemic mixture” and “racemate”refer to an equimolar mixture of two enantiomeric species, devoid ofoptical activity.

The term “tautomer” or “tautomeric form” refers to structural isomers ofdifferent energies that are interconvertible via a low energy barrier.For example, proton tautomers (also known as prototropic tautomers)include interconversions via migration of a proton, such as keto-enoland imine-enamine isomerizations. Valence tautomers includeinterconversions by reorganization of some of the bonding electrons.

The term “imine reactive reagent” refers to a reagent that is capable ofreacting with an imine group. Examples of imine reactive reagentincludes, but is not limited to, sulfites (H₂SO₃, H₂SO₂ or a salt ofHSO₃ ⁻, SO₃ ²⁻ or HSO₂ ⁻ formed with a cation), metabisulfite (H₂S₂O₅ ora salt of S₂O₅ ²⁻ formed with a cation), mono, di, tri, andtetra-thiophosphates (PO₃SH₃, PO₂S₂H₃, POS₃H₃, PS₄H₃ or a salt ofPO₃S³⁻, PO₂S₂ ³⁻, POS₃ ³⁻ or PS₄ ³⁻ formed with a cation), thiophosphate esters ((R^(i)O)₂PS(OR^(i)), R^(i)SH, R^(i)SOH, R^(i)SO₂H,R^(i)SO₃H), various amines (hydroxyl amine (e.g., NH₂OH), hydrazine(e.g., NH₂NH₂), NH₂O—R^(i), R^(i)′NH—R^(i), NH₂—R^(i)), NH₂—CO—NH₂,NH₂—C(═S)—NH_(2′) thiosulfate (H₂S₂O₃ or a salt of S₂O₃ ²⁻ formed with acation), dithionite (H₂S₂O₄ or a salt of S₂O₄ ²⁻ formed with a cation),phosphorodithioate (P(═S)(OR^(k))(SH)(OH) or a salt thereof formed witha cation), hydroxamic acid (R^(k)C(═O)NHOH or a salt formed with acation), hydrazide (R^(k)CONHNH₂), formaldehyde sulfoxylate (HOCH₂SO₂Hor a salt of HOCH₂SO₂ ⁻ formed with a cation, such as HOCH₂SO₂ ⁻Na⁺),glycated nucleotide (such as GDP-mannose), fludarabine or a mixturethereof, wherein R^(i) and R^(i′) are each independently a linear orbranched alkyl having 1 to 10 carbon atoms and are substituted with atleast one substituent selected from —N(R^(j))₂, —CO₂H, —SO₃H, and —PO₃H;R^(i) and R^(i′) can be further optionally substituted with asubstituent for an alkyl described herein; R^(j) is a linear or branchedalkyl having 1 to 6 carbon atoms; and R^(k) is a linear, branched orcyclic alkyl, alkenyl or alkynyl having 1 to 10 carbon atoms, aryl,heterocyclyl or heteroaryl (preferably, R^(k) is a linear or branchedalkyl having 1 to 4 carbon atoms; more preferably, R^(k) is methyl,ethyl or propyl). Preferably, the cation is a monovalent cation, such asNa⁺ or K⁺. Preferably, the imine reactive reagent is selected fromsulfites, hydroxyl amine, urea and hydrazine. More preferably, the iminereactive reagent is NaHSO₃ or KHSO₃.

The phrase “pharmaceutically acceptable salt” as used herein, refers topharmaceutically acceptable organic or inorganic salts of a compound ofthe invention. Exemplary salts include, but are not limited, to sulfate,citrate, acetate, oxalate, chloride, bromide, iodide, nitrate,bisulfate, phosphate, acid phosphate, isonicotinate, lactate,salicylate, acid citrate, tartrate, oleate, tannate, pantothenate,bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate,gluconate, glucuronate, saccharate, formate, benzoate, glutamate,methanesulfonate “mesylate,” ethanesulfonate, benzenesulfonate,p-toluenesulfonate, pamoate (i.e.,1,1′-methylene-bis-(2-hydroxy-3-naphthoate)) salts, alkali metal (e.g.,sodium and potassium) salts, alkaline earth metal (e.g., magnesium)salts, and ammonium salts. A pharmaceutically acceptable salt caninvolve the inclusion of another molecule such as an acetate ion, asuccinate ion or other counter ion. The counter ion can be any organicor inorganic moiety that stabilizes the charge on the parent compound.Furthermore, a pharmaceutically acceptable salt can have more than onecharged atom in its structure. Instances where multiple charged atomsare part of the pharmaceutically acceptable salt can have multiplecounter ions. Hence, a pharmaceutically acceptable salt can have one ormore charged atoms and/or one or more counter ion.

If the compound of the invention is a base, the desired pharmaceuticallyacceptable salt can be prepared by any suitable method available in theart, for example, treatment of the free base with an inorganic acid,such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,methanesulfonic acid, phosphoric acid and the like, or with an organicacid, such as acetic acid, maleic acid, succinic acid, mandelic acid,fumaric acid, malonic acid, pyruvic acid, oxalic acid, glycolic acid,salicylic acid, a pyranosidyl acid, such as glucuronic acid orgalacturonic acid, an alpha hydroxy acid, such as citric acid ortartaric acid, an amino acid, such as aspartic acid or glutamic acid, anaromatic acid, such as benzoic acid or cinnamic acid, a sulfonic acid,such as p-toluenesulfonic acid or ethanesulfonic acid, or the like.

If the compound of the invention is an acid, the desiredpharmaceutically acceptable salt can be prepared by any suitable method,for example, treatment of the free acid with an inorganic or organicbase, such as an amine (primary, secondary or tertiary), an alkali metalhydroxide or alkaline earth metal hydroxide, or the like. Illustrativeexamples of suitable salts include, but are not limited to, organicsalts derived from amino acids, such as glycine and arginine, ammonia,primary, secondary, and tertiary amines, and cyclic amines, such aspiperidine, morpholine and piperazine, and inorganic salts derived fromsodium, calcium, potassium, magnesium, manganese, iron, copper, zinc,aluminum and lithium.

As used herein, the term “solvate” means a compound that furtherincludes a stoichiometric or non-stoichiometric amount of solvent suchas water, isopropanol, acetone, ethanol, methanol, DMSO, ethyl acetate,acetic acid, and ethanolamine dichloromethane, 2-propanol, or the like,bound by non-covalent intermolecular forces. Solvates or hydrates of thecompounds are readily prepared by addition of at least one molarequivalent of a hydroxylic solvent such as methanol, ethanol,1-propanol, 2-propanol or water to the compound to result in solvationor hydration of the imine moiety.

A “metabolite” or “catabolite” is a product produced through metabolismor catabolism in the body of a specified compound, a derivative thereof,or a conjugate thereof, or salt thereof. Metabolites of a compound, aderivative thereof, or a conjugate thereof, can be identified usingroutine techniques known in the art and their activities determinedusing tests such as those described herein. Such products can result forexample from the oxidation, hydroxylation, reduction, hydrolysis,amidation, deamidation, esterification, deesterification, enzymaticcleavage, and the like, of the administered compound. Accordingly, theinvention includes metabolites of compounds, a derivative thereof, or aconjugate thereof, of the invention, including compounds, a derivativethereof, or a conjugate thereof, produced by a process comprisingcontacting a compound, a derivative thereof, or a conjugate thereof, ofthis invention with a mammal for a period of time sufficient to yield ametabolic product thereof.

The phrase “pharmaceutically acceptable” indicates that the substance orcomposition must be compatible chemically and/or toxicologically, withthe other ingredients comprising a formulation, and/or the mammal beingtreated therewith.

The term “protecting group” or “protecting moiety” refers to asubstituent that is commonly employed to block or protect a particularfunctionality while reacting other functional groups on the compound, aderivative thereof, or a conjugate thereof. For example, an“amine-protecting group” or an “amino-protecting moiety” is asubstituent attached to an amino group that blocks or protects the aminofunctionality in the compound. Such groups are well known in the art(see for example P. Wuts and T. Greene, 2007, Protective Groups inOrganic Synthesis, Chapter 7, J. Wiley & Sons, NJ) and exemplified bycarbamates such as methyl and ethyl carbamate, FMOC, substituted ethylcarbamates, carbamates cleaved by 1,6-β-elimination (also termed “selfimmolative”), ureas, amides, peptides, alkyl and aryl derivatives.Suitable amino-protecting groups include acetyl, trifluoroacetyl,t-butoxycarbonyl (BOC), benzyloxycarbonyl (CBZ) and9-fluorenylmethylenoxycarbonyl (Fmoc). For a general description ofprotecting groups and their use, see P. G. M. Wuts & T. W. Greene,Protective Groups in Organic Synthesis, John Wiley & Sons, New York,2007.

The term “amino acid” refers to naturally occurring amino acids ornon-naturally occurring amino acid. In one embodiment, the amino acid isrepresented by NH₂—C(R^(aa′)R^(aa))—C(═O)OH, wherein R^(aa) and R^(aa′)are each independently H, an optionally substituted linear, branched orcyclic alkyl, alkenyl or alkynyl having 1 to 10 carbon atoms, aryl,heteroaryl or heterocyclyl or R^(aa) and the N-terminal nitrogen atomcan together form a heterocyclic ring (e.g., as in proline). The term“amino acid residue” refers to the corresponding residue when onehydrogen atom is removed from the amine and/or carboxy end of the aminoacid, such as —NH—C(R^(aa′)R^(aa))—C(═O)O—.

The term “cation” refers to an ion with positive charge. The cation canbe monovalent (e.g., Na⁺, K⁺, NH₄ ⁺ etc.), bi-valent (e.g., Ca²⁺, Me²⁺,etc.) or multi-valent (e.g., Al³⁺ etc.). Preferably, the cation ismonovalent.

The term “reactive ester group” refers to a group an ester group thatcan readily react with an amine group to form amide bond. Exemplaryreactive ester groups include, but are not limited to,N-hydroxysuccinimide esters, N-hydroxyphthalimide esters, N-hydroxysulfo-succinimide esters, para-nitrophenyl esters, dinitrophenyl esters,pentafluorophenyl esters and their derivatives, wherein said derivativesfacilitate amide bond formation. In certain embodiments, the reactiveester group is a N-hydroxysuccinimide ester or a N-hydroxysulfo-succinimide ester.

The term “amine reactive group” refers to a group that can react with anamine group to form a covalent bond. Exemplary amine reactive groupsinclude, but are not limited to, reactive ester groups, acyl halides,sulfonyl halide, imidoester, or a reactive thioester groups. In certainembodiments, the amine reactive group is a reactive ester group. In oneembodiment, the amine reactive group is a N-hydroxysuccinimide ester ora N-hydroxy sulfo-succinimide ester.

The term “thiol-reactive group” refers to a group that can react with athiol (—SH) group to form a covalent bond. Exemplary thiol-reactivegroups include, but are not limited to, maleimide, haloacetyl,aloacetamide, vinyl sulfone, vinyl sulfonamide or vinyal pyridine. Inone embodiment, the thiol-reactive group is maleimide.

As used in the present disclosure and claims, the singular forms “a,”“an,” and “the” include plural forms unless the context clearly dictatesotherwise.

It is understood that wherever embodiments are described herein with thelanguage “comprising,” otherwise analogous embodiments described interms of “consisting of” and/or “consisting essentially of” are alsoprovided.

The term “and/or” as used in a phrase such as “A and/or B” herein isintended to include both “A and B,” “A or B,” “A,” and “B.” Likewise,the term “and/or” as used in a phrase such as “A, B, and/or C” isintended to encompass each of the following embodiments: A, B, and C; A,B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B(alone); and C (alone).

Antibodies, Compounds, and Immunoconjugates Nomenclature

As used herein, the nomenclature used for the anti-CD123 antibodies,cytotoxic compounds, and their immunoconjugates generally adopt thefollowing meanings.

CD123-3, -6, and -14 (or CD123 Mu-3, -6, and -14; or muCD123-3, -6, and14) are three murine anti-CD123 monoclonal antibodies. The CDR1-3sequences of the heavy and light chains (VH-CDR1-3, and VL-CDR1-3) areprovided in Tables 1 and 2, with the associated SEQ ID NOs: 1-25. Theheavy chain variable region (HCVR) sequences are provided in Table 3Awith the associated SEQ ID NOs: 26, 28, and 30. Their light chainvariable region (LCVR) sequences are provided in Table 4A with theassociated SEQ ID NOs: 27, 29, and 31. The full length heavy chain (HC)sequences of the murine antibodies are provided in Table 5 (SEQ ID NOs:42, 44, and 46), and the full length light chain (LC) sequences of themurine antibodies are provided in Table 6 (SEQ ID NOs: 43, 45, and 47).

chCD123-3, -6, and -14 are the corresponding murine-human chimericantibodies having the murine heavy and light chain variable regions andthe human constant region sequences. For example, the chimeric antibodychCD123-6 is comprised of the mouse HCVR and LCVR of SEQ ID NOs: 28 and29, respectively, together with the human IgG1 and Kappa constantsequences for the heavy and light chains, respectively. See Example 3.

huCD123-3, -6, and -14 are the corresponding humanized antibodies. Whenthe humanization is by way of CDR grafting of the 6 corresponding murineCDR regions (HC and LC CDR1-3), the letter “G” immediately follows theclone designation, which is in turn followed by a version number thatdesignates the origin of the human light chain and heavy chain variableregion sequences. Thus huCD123-6Gv4.6 refers to the humanized CD123antibody based on grafting (“G”) the 6 CDR regions from thecorresponding muCDR123-6 antibody, onto the human light chain variableregion Gv4 and the heavy chain variable region Gv6. Similarly, -Gv4.7comprises human light chain variable region Gv4 and heavy chain variableregion Gv7; and -Gv1.1 comprises human light chain variable region Gv1and heavy chain variable region Gv1.

The three HCVR sequences, huCD123-6Gv1, -Gv6, and -Gv7 are provided inTable 3A (SEQ ID NOs: 32 and 34, with SEQ ID NO: 34 representing both-Gv6 and -Gv7 since they differ only at the 2^(nd) residue Xaa), andtheir DNA coding sequences in Table 3B (SEQ ID NOs: 62, 64, and 66). Thethree full length HC sequences, huCD123-6Gv1, -Gv6, and -Gv7 areprovided in Table 5 (SEQ ID NOs: 48 and 50, with SEQ ID NO: 50representing both full length -Gv6 and -Gv7 since they differ only atthe 2^(nd) residue Xaa).

The two LCVR sequences, huCD123-6Gv1 and -Gv4, are provided in Table 4A(SEQ ID NOs: 33 and 35, and their DNA coding sequences in Table 4B (SEQID NOs: 63, and 65). The two full length LC sequences, huCD123-6Gv1 and-Gv4, are provided in Table 6 (SEQ ID NOs: 49 and 51).

When humanization is by way of resurfacing, the resurfaced heavy chainsequences are designated by “rh” immediately following the murine CD123antibody clone number, and are further designated by one of two versionof the resurfaced sequences, v1.0 or v1.1. Thus huCD123-6rhv1.0 and-rhv1.1 are resurfaced heavy chain sequences with CDR regionscorresponding to the muCD123-6 antibody, with version designation of 1.0and 1.1 respectively. See HCVR SEQ ID NOs: 39 and 40 in Table 3A, andSEQ ID NOs: 68 and 69 in Table 3B. Also see full length HC SEQ ID NOs:59 and 60 in Table 5.

Likewise, the only version of the resurfaced light chain sequence,huCD123-6rlv1.0, has LCVR SEQ ID NO: 41 in Table 4A, and full length LCSEQ ID NO: 61 in Table 6.

A resurfaced antibody having huCD123-6rhv1.0 and huCD123-6rlv1.0 ishuCD123-6Rv1.0; and a resurfaced antibody having huCD123-6rhv1.1 andhuCD123-6rlv1.0 is huCD123-6Rv1.1.

NTS2 or “S2” for short refers to an antibody having an engineered Ser atthe N-terminus of heavy chain. The S2 variant of the huCD123-6Gv6/7 hasHCVR sequence SEQ ID NO: 38 in Table 3A, and full length HC proteinsequence SEQ ID NO: 53 in Table 5.

Likewise, NTS3 or “S3” for short refers to an antibody having anengineered Ser at the N-terminus of light chain. The S3 variant of thehuCD123-6Gv4 has LCVR sequence SEQ ID NO: 37 in Table 4A, and fulllength LC protein sequence SEQ ID NO: 58 in Table 6.

An antibody comprising an engineered N-terminal Ser (either S2 or S3)may be conjugated with a cytotoxic drug/agent through either theoxidized N-terminal Ser, or through the “conventional” Lys linkage. Ifthe drug linkage is through oxidized N-terminal Ser, the conjugate namecontains a “SeriMab” designation. If the drug linkage is through Lys,then the conjugate name does not contain SeriMab (despite the fact thatthere is an S2 or S3 designation to signal the presence of engineeredSer at the N-terminus). The particular linkage type will also beapparent based on the cytotoxin reactive group. For example,huCD123-6Gv4.7S3-SeriMab-D8 refers to conjugate between D8 and thehumanized CD123 antibody huCD123-6Gv4.7S3, through the oxidizedN-terminal Ser on the light chain. The humanized CD123 antibody has thegrafted murine CD123-6 CDR regions, the human LC Gv4 and heavy chainGv7, and the N-terminal of the light chain has an engineered Ser (S3).In contrast, huCD123-6Gv4.7S3-sSPDB-D1 refers to conjugate between D1and the same humanized CD123 antibody huCD123-6Gv4.7S3, through Lyslinkage via a sulfonated SPDB linker.

In certain embodiments, if both the light chain and heavy chainN-termini contain the engineered Ser, “S2S3” or “S2S3-SeriMab” mayappear in the antibody name.

Certain antibodies of the invention have an engineered Cys in the heavychain CH3 domain, at a position corresponding to the same Kabat positionof the 5^(th) to the last Cys in SEQ ID NO: 54. Such HCs or antibodiescomprising such HCs contain the designation CysMab. ThushuCD123-6Gv4.6-CysMab is a humanized CD123 antibody that has graftedmuCD123-6 CDR regions, is based on the human light chain Gv4 and heavychain Gv6 sequences, wherein an engineered Cys is located in the HC CH3region at a position corresponding to the 5^(th) to the last Cys in SEQID NO: 54. Similarly, its heavy chain sequence is huCD123-6Gv6-CysMab.In addition, huCD123-6Gv4.6S2-CysMab is otherwise identical, but has anengineered Ser at the N-terminus of the heavy chain, and its heavy chainsequence is huCD123-6Gv6S2-CysMab.

The resurfaced antibody described above may be further engineered tocontain N-terminal Ser at either the light chain (S3 variant of theresurfaced antibody) or the heavy chain (S2 variant of the resurfacedantibody), or both (see below). Alternatively or in addition, theresurfaced antibody may have an engineered Cys in the heavy chain CH3domain at a position corresponding to the same Kabat position of the5^(th) to the last Cys of SEQ ID NO: 54 (the CysMab version of theresurfaced antibody). A resurfaced antibody can have both engineered Cysand N-terminal Ser.

In the conjugates formed between such CysMab and cytotoxin, however, atleast in theory, the cytotoxin can be linked to the CysMab eitherthrough the Cys or through the conventional Lys. As used herein,however, without specific indication, a conjugate with a CysMabdesignation refers to a conjugate between a CysMab and a cytotoxinthough the Cys-linkage (not the Lys linkage). The particular linkagetype will also be apparent based on the cytotoxin reactive group.

Other variations or combinations of the above general nomenclature arealso contemplated and will be readily apparent to one of skill in theart. For example, huCD123-6Rv1.1-CysMab is the resurfaced version ofhuCD123-6 (v1.1) that has an engineered Cys located in the HC CH3 regionat a position corresponding to the 5^(th) to the last Cys in SEQ ID NO:54.

The antibodies or antigen-binding fragments thereof of the invention maybe conjugated to certain cytotoxic agents, either through linkage withthe Lys side chain amino group, the Cys side chain thiol group, or anoxidized N-terminal Ser/Thr. Certain representative (non-limiting)cytotoxic agents described in the specification (including the examples)are listed below for illustration purpose. Note that most compounds suchas D1, D2, D4, DGN462, D3, D6, etc., may be sulfonated (not shown here,but see FIG. 17 compound sD1, FIG. 15 compound sDGN462, and the FIG. 16compound sD8) at one of the indolinobenzodiazepine monomers in certainexamples. For compound D5′, both indolinobenzodiazepine monomers may besulfonated.

Com- pound No. Structure D1

D2

DGN462

D3

D4

D5

D5′

D6

D7

D8

D9

Note that several agents only differ slightly due to the differentlinkage chemistry required for linking the cytotoxin to differentantibody side chains (i.e., Lys-linkage, Cys-linkage, oxidizedN-terminal Ser linkage). Nevertheless, these related cytotoxins aregiven different “D” designations. See D1 and D4, as well as D2, D5, andD8.

Conjugates of the subject antibodies and the cytotoxic agents generallyfollow the nomenclature of the antibodies and cytotoxic agents asdescribed above.

For example, huCD123-6Gv4.6-sulfo-SPDB-D1 is a conjugate of thehuCD123-6Gv4.6 antibody to compound D1 through a sulfonated SPDB linker,at one or more Lys residues of the antibody. huCD123-6-CX1-1-DM1 is aconjugate of the huCD123-6 antibody conjugated with the cytotoxic agentDM1 via a triglycyl linker named “CX1-1 linker,” at one or more of theLys residues of the antibody. See Example 9e.

One notable exception is the conjugate huCD123-6-SeriMab-sD1 shown inFIGS. 7B and 17, in which the short linker sequence between thehuCD123-6-SeriMab and the sD1 cytotoxin is not explicitly recited in theconjugate name. Similarly, conjugate huCD123-6-SeriMab-sDGN462 in FIG.15 is also an exception to the general rules above.

2. CD123-Binding Agents

In a first aspect, the present invention provides agents thatspecifically bind CD123/IL-3Rα, such as human CD123/IL-3Rα. These agentsare generally referred to herein as “CD123/IL-3Rα-binding agents.” Incertain embodiments, the CD123/IL-3Rα-binding agents are antibodies orantigen-binding fragments thereof (or “antibodies” for simplicity),immunoconjugates thereof or polypeptides thereof.

The amino acid and nucleotide sequences for human and other species ofCD123/IL-3Rα are known in the art. For example, the human CD123/IL-3Rαsplicing variant 1 protein sequence as depicted in NCBI RefSeq NP_002174is reproduced below:

(SEQ ID NO: 36) 1MVLLWLTLLL IALPCLLQTK EDPNPPITNL RMKAKAQQLT WDLNRNVTDI ECVKDADYSM 61PAVNNSYCQF GAISLCEVTN YTVRVANPPF STWILFPENS GKPWAGAENL TCWIHDVDFL 121SCSWAVGPGA PADVQYDLYL NVANRRQQYE CLHYKTDAQG TRIGCRFDDI SRLSSGSQSS 181HILVRGRSAA FGIPCTDKFV VFSQIEILTP PNMTAKCNKT HSFMHWKMRS HFNRKFRYEL 241QIQKRMQPVI TEQVRDRTSF QLLNPGTYTV QIRARERVYE FLSAWSTPQR FECDQEEGAN 301TRAWRTSLLI ALGTLLALVC VFVICRRYLV MQRLFPRIPH MKDPIGDSFQ NDKLVVWEAG 361KAGLEECLVT EVQVVQKT

The above sequence shows the CD123/IL-3R alpha precursor chain protein,which is composed by 378 amino acids, containing the extracellulardomain (residues 1-306, including an 18-residue N-terminal signalpeptide), a 20 amino acid transmembrane domain, and a short cytoplasmictail of 52 amino acids.

The human CD123/IL-3Rα splicing variant 1 nucleic acid sequence asdepicted in NCBI RefSeq NM_002183 is reproduced below:

(SEQ ID NO: 52) 1GTCAGGTTCA TGGTTACGAA GCTGCTGACC CCAGGATCCC AGCCCGTGGG AGAGAAGGGG 61GTCTCTGACA GCCCCCACCC CTCCCCACTG CCAGATCCTT ATTGGGTCTG AGTTTCAGGG 121GTGGGGCCCC AGCTGGAGGT TATAAAACAG CTCAATCGGG GAGTACAACC TTCGGTTTCT 181CTTCGGGGAA AGCTGCTTTC AGCGCACACG GGAAGATATC AGAAACATCC TAGGATCAGG 241ACACCCCAGA TCTTCTCAAC TGGAACCACG AAGGCTGTTT CTTCCACACA GTACTTTGAT 301CTCCATTTAA GCAGGCACCT CTGTCCTGCG TTCCGGAGCT GCGTTCCCGA TGGTCCTCCT 361TTGGCTCACG CTGCTCCTGA TCGCCCTGCC CTGTCTCCTG CAAACGAAGG AAGATCCAAA 421CCCACCAATC ACGAACCTAA GGATGAAAGC AAAGGCTCAG CAGTTGACCT GGGACCTTAA 481CAGAAATGTG ACCGATATCG AGTGTGTTAA AGACGCCGAC TATTCTATGC CGGCAGTGAA 541CAATAGCTAT TGCCAGTTTG GAGCAATTTC CTTATGTGAA GTGACCAACT ACACCGTCCG 601AGTGGCCAAC CCACCATTCT CCACGTGGAT CCTCTTCCCT GAGAACAGTG GGAAGCCTTG 661GGCAGGTGCG GAGAATCTGA CCTGCTGGAT TCATGACGTG GATTTCTTGA GCTGCAGCTG 721GGCGGTAGGC CCGGGGGCCC CCGCGGACGT CCAGTACGAC CTGTACTTGA ACGTTGCCAA 781CAGGCGTCAA CAGTACGAGT GTCTTCACTA CAAAACGGAT GCTCAGGGAA CACGTATCGG 841GTGTCGTTTC GATGACATCT CTCGACTCTC CAGCGGTTCT CAAAGTTCCC ACATCCTGGT 901GCGGGGCAGG AGCGCAGCCT TCGGTATCCC CTGCACAGAT AAGTTTGTCG TCTTTTCACA 961GATTGAGATA TTAACTCCAC CCAACATGAC TGCAAAGTGT AATAAGACAC ATTCCTTTAT 1021GCACTGGAAA ATGAGAAGTC ATTTCAATCG CAAATTTCGC TATGAGCTTC AGATACAAAA 1081GAGAATGCAG CCTGTAATCA CAGAACAGGT CAGAGACAGA ACCTCCTTCC AGCTACTCAA 1141TCCTGGAACG TACACAGTAC AAATAAGAGC CCGGGAAAGA GTGTATGAAT TCTTGAGCGC 1201CTGGAGCACC CCCCAGCGCT TCGAGTGCGA CCAGGAGGAG GGCGCAAACA CACGTGCCTG 1261GCGGACGTCG CTGCTGATCG CGCTGGGGAC GCTGCTGGCC CTGGTCTGTG TCTTCGTGAT 1321CTGCAGAAGG TATCTGGTGA TGCAGAGACT CTTTCCCCGC ATCCCTCACA TGAAAGACCC 1381CATCGGTGAC AGCTTCCAAA ACGACAAGCT GGTGGTCTGG GAGGCGGGCA AAGCCGGCCT 1441GGAGGAGTGT CTGGTGACTG AAGTACAGGT CGTGCAGAAA ACTTGAGACT GGGGTTCAGG 1501GCTTGTGGGG GTCTGCCTCA ATCTCCCTGG CCGGGCCAGG CGCCTGCACA GACTGGCTGC 1561TGGACCTGCG CACGCAGCCC AGGAATGGAC ATTCCTAACG GGTGGTGGGC ATGGGAGATG 1621CCTGTGTAAT TTCGTCCGAA GCTGCCAGGA AGAAGAACAG AACTTTGTGT GTTTATTTCA 1681TGATAAAGTG ATTTTTTTTT TTTTAACCCA AAA

Proteins and nucleic acid sequences of CD123/IL-3Rα from other non-humanspecies can be readily retrieved from public database such as GenBank,using sequence search tools known in the art (such as NCBI BLASTp orBLASTn) and the above protein and nucleic acid sequences as querysequences, respectively.

Such sequences from the non-human species can be aligned with the humansequences using any of many art-recognized sequence alignment tools,such as those described herein and above, such that any amino acidresidues or nucleotides “corresponding to” any given human sequences orregions of sequences can be readily obtained.

Thus, one aspect of the invention provides an antibody orantigen-binding fragment thereof that: (a) binds an epitope within aminoacids 101 to 346 of human CD123 antigen, and (b) inhibits IL3-dependentproliferation in antigen-positive TF-1 cells.

In some embodiments, an anti-CD123/IL-3Rα antibody or antigen-bindingfragment thereof can specifically binds to an epitope of SEQ ID NO: 36.In certain embodiments, the epitope is within a region corresponding toresidues 101-346 of human CD123/IL-3Rα. In certain embodiments, theepitope is within a region corresponding to residues 101-204 of SEQ IDNO: 36. In certain other embodiments, the epitope is within a regioncorresponding to residues 205-346 of SEQ ID NO: 36. In certainembodiments, the epitope is not within a region corresponding toresidues 1-100 of human CD123/IL-3Rα.

In certain embodiments, the CD123/IL-3Rα-binding agents (e.g.,antibodies) inhibit IL3-dependent signaling, such as IL-3-dependentproliferation of CD123-positive TF-1 cells. While not wishing to bebound by any particular theory, the CD123/IL-3Rα-binding agents (e.g.,antibodies) of the invention bind to CD123, such as within a CD123region corresponding to residues 101-346 (e.g., residues 101-204, or205-346) of human CD123/IL-3Rα, and prevents, reduces, diminishes, orotherwise inhibits productive binding between CD123 and the IL-3 ligand,and/or productive binding between CD123 and the common beta chain CD131,leading to reduced or abolished IL-3 dependent signaling.

In a related aspect, the invention provides an antibody orantigen-binding fragment thereof that: (a) binds an epitope within aminoacids 1 to 100 of human CD123 antigen, and (b) inhibits IL3-dependentproliferation in antigen-positive TF-1 cells, with an IC₅₀ value of 0.1nM or less (e.g., 0.08 nM, 0.05 nM, 0.03 nM).

In certain embodiments, binding by the CD123/IL-3Rα-binding agents(e.g., antibodies) of the invention inhibits (e.g., preferentiallyinhibits) the proliferation of leukemic stem cells (LSCs), leukemiaprogenitors (LPs), or leukemic blasts, but do not substantially inhibitthe proliferation of the normal hematopoietic stem cells (HSCs).

Inhibition of cell proliferation can be conducted using any standardassays known in the art, including but are not limited to flowcytometry. For example, normal HSCs, LSCs, LPs, and leukemia blasts canbe separated using flow cytometry based on the difference on expressionof cell surface markers, and the relative number of the surviving orremaining cells, after incubating with the testing agents, can bequantitatively measured and compared.

Inhibition of cell proliferation of the LSCs, LPs, or leukemia blasts ascompared to normal HSCs can also be assayed using in vitro potency assayon primary cancer cells, such as primary AML cells. For example, AMLcells (or normal human bone marrow samples containing normal HSCs) canbe exposed to various concentrations of the subject anti-CD123antibodies, antigen-binding fragments thereof, immuno-conjugatesthereof, or polypeptide comprising the antibodies or antigen-bindingfragments for 24 hrs. Non-targeting (isotype-matched) antibodies, orimmuno-conjugates (ADC) control can also be used in the assay. Samplescan be divided into a short-term liquid culture (STLC) assay to measurethe cytotoxicity toward the LSCs, LPs, or leukemia blasts; and along-term liquid culture (LTLC) assay to measure the effect on the LSCsand normal HSCs. STLC can be used to measure colony forming units 10-14days in cells, e.g., following plating in semi-solid MethoCult H4230medium (Stemcell technologies). The LTLC assays can be performedsimilarly with the addition of growth factors for long-term culture 5-7weeks. In both assays, colonies can be counted to determine colonyforming units per number of cells initially plated. LTLC colonies can befurther analyzed for the presence of cancer (e.g., AML) molecularmarkers using PCR or FISH or both.

In certain embodiments, the antibody or antigen-binding fragment thereofbinds to human CD123 antigen-positive cells with a dissociation constant(K_(d)) of 0.3 nM or lower. In certain embodiments, the antibodies orantigen-binding fragments thereof bind to human CD123 with a K_(d)between 0.05 and 0.3 nM, or between 0.05 and 0.2 nM, or between 0.05 and0.1 nM, or between 0.01 nM and 0.3 nM, or between 0.01 nM and 0.2 nM, orbetween 0.01 nM and 0.1 nM.

In certain embodiments, the antibodies or antigen-binding fragmentsthereof bind to cynomolgus monkey CD123. In certain embodiments, theantibodies or antigen-binding fragments thereof bind to cynomolgusmonkey CD123 with a K_(d) between 0.05 and 0.3 nM, or between 0.05 and0.2 nM, or between 0.05 and 0.1 nM.

In certain embodiments, the antibodies or antigen-binding fragmentsthereof bind both human and cynomolgus monkey CD123 with a substantiallysimilar binding affinity. In certain embodiments, the antibodies orantigen-binding fragments thereof bind to both human and cynomolgusmonkey CD123 with K_(d) between 0.05 and 0.3 nM, or between 0.05 and 0.2nM, or between 0.05 and 0.1 nM.

In certain embodiments, the K_(d) value is based on cell-based bindingassay. In certain embodiments, the K_(d) value is measured by flowcytometry. In certain embodiments, the K_(d) value is measured bysurface plasmon resonance (such as by using the BIOCORE™ surface plasmonresonance system). In certain embodiments, the K_(d) value is measuredby radioimmunoassay (RIA). In certain embodiments, the K_(d) is measuredby any other art-recognized methods.

In certain embodiments, the antibody or antigen-binding fragment thereofinhibits at least 50% of IL3-dependent proliferation in antigen-positiveTF-1 cells at a concentration of 0.5 nM or lower.

In certain embodiments, the CD123/IL-3Rα-binding agents are CD123/IL-3Rαantibodies or antigen-binding fragments thereof that comprise a heavychain variable region (HCVR) and a light chain variable region (LCVR),each comprising three CDR regions (e.g., CDR1-CDR3 for the HCVR, andCDR1-CDR3 for the LCVR), wherein the composite CDRs for the HCVR andLCVR are any of the sequences provided in Tables 1 and 2 below.

TABLE 1 Heavy Chain Variable Region CDR Amino Acid Sequences AntibodyAlt Name VH-CDR1 VH-CDR2 VH-CDR3 CD123-3 CD123Mu-3 SYVMH YIKPYKDGTKEGENGYYDAMDY (SEQ ID (SEQ ID NO: 2) (SEQ ID NO: 4) NO: 1)YIKPYKDGTKYNEKFKG (Kabat)(SEQ ID NO: 3) CD123-6 CD123Mu-6 SSIMHYIKPYNDGTK EGGNDYYDTMDY (SEQ ID Murine + Grafted (SEQ ID NO: 11) NO: 5)(SEQ ID NO: 6) YIRPYNDGTR (resurfaced version 1.0) (SEQ ID NO: 7)YIKPYNDGTKYNEKFKG (Kabat Murine + Grafted) (SEQ ID NO: 8)YIRPYNDGTRYNQKFQG (Kabat - resurfaced v1.0) (SEQ ID NO: 9)YIKPYNDGTKYNQKFQG (Kabat - resurfaced v1.1) (SEQ ID NO: 10) CD123-14CD123Mu-14 NYAMS TINSGGSFTY QSEAYYGYDKRT (SEQ ID (SEQ ID NO: 13)(SEQ ID NO: 15) NO: 12) QSEAYYGYDKRTW FAY (SEQ ID NO: 70)TINSGGSFTYYPDSVKG (Kabat) (SEQ ID NO: 14)

TABLE 2 Light Chain Variable Region CDR Amino Acid Sequences AntibodyAlt Name VL-CDR1 VL-CDR2 VL-CDR3 CD123-3 CD123Mu-3 KASQDINKYIA YTSTLQPLQYDNLLYT (SEQ ID NO: 16) (SEQ ID NO: 17) (SEQ ID NO: 18) CD123-6CD123Mu-6 KASQDINSYLS RVNRLVD LQYDAFPYT (SEQ ID NO: 19) (SEQ ID NO: 21)(SEQ ID NO: 22) RASQDINSYLS RVNRLVS Humanized (SEQ ID NO: 71)(SEQ ID NO: 20) RASQDINSYLA (SEQ ID NO: 72) CD123-14 CD123Mu-14RASQSVGTSIH YASESIS QQSKSWPLT (SEQ ID NO: 23) (SEQ ID NO: 24)(SEQ ID NO: 25)

In certain embodiments, the anti-CD123/IL-3Rα antibodies andantigen-binding fragments thereof comprise: a) at least one heavy chainvariable region or fragment thereof comprising three sequentialcomplementarity-determining regions (CDR) CDR1, CDR2, and CDR3,respectively, wherein, with the exception of 1, 2, or 3 conservativeamino acid substitutions, CDR1 is selected from the group consisting of:SEQ ID NOs: 1, 5, and 12, CDR2 is selected from the group consisting of:SEQ ID NOs: 2-3, 6-10, and 13-14, and, optionally, CDR3 is selected fromthe group consisting of: SEQ ID NOs: 4, 11, 15 and 70; and b) at leastone light chain variable region or fragment thereof comprising threesequential complementarity-determining regions (CDR) CDR1, CDR2, andCDR3, respectively, wherein, with the exception of 1, 2, or 3conservative amino acid substitutions, CDR1 is selected from the groupconsisting of: SEQ ID NOs: 16, 19-20, 23 and 72, CDR2 is selected fromthe group consisting of: SEQ ID NOs: 17, 21, 24 and 71, and, optionally,CDR3 is selected from the group consisting of: SEQ ID NOs: 18, 22, and25.

In certain embodiments, the anti-CD123/IL-3Rα antibodies andantigen-binding fragments thereof comprise: a) at least one heavy chainvariable region or fragment thereof comprising three sequentialcomplementarity-determining regions (CDR) CDR1, CDR2, and CDR3,respectively, wherein, with the exception of 1, 2, or 3 conservativeamino acid substitutions, CDR1 is selected from the group consisting of:SEQ ID NOs: 1, 5, and 12, CDR2 is selected from the group consisting of:SEQ ID NOs: 2-3, 6-10, and 13-14, and, optionally, CDR3 is selected fromthe group consisting of: SEQ ID NOs: 4, 11, and 15; and b) at least onelight chain variable region or fragment thereof comprising threesequential complementarity-determining regions (CDR) CDR1, CDR2, andCDR3, respectively, wherein, with the exception of 1, 2, or 3conservative amino acid substitutions, CDR1 is selected from the groupconsisting of: SEQ ID NOs: 16, 19-20, and 23, CDR2 is selected from thegroup consisting of: SEQ ID NOs: 17, 21, and 24, and, optionally, CDR3is selected from the group consisting of: SEQ ID NOs: 18, 22, and 25.

In certain embodiments, the conservative amino acid substitutionscomprise a substitution of a Lys in a CDR by an Arg (such as theLys-to-Arg substitutions in SEQ ID NOs: 6 and 7, 8 and 9, and 19 and20). In certain embodiments, the antibody is a CDR-grafted humanizedantibody comprising mouse CDR regions, and wherein one or more (e.g., 1,2, 3, 4, 5, 6, 7, or 8) heavy chain and/or light chain framework regionvernier zone residues of the antibody is of mouse origin.

In certain embodiments, the anti-CD123/IL-3Rα antibodies andantigen-binding fragments thereof comprise: a) an immunoglobulin heavychain variable region comprising a CDR1 having an amino acid sequenceset forth in SEQ ID NO: 1, a CDR2 having an amino acid sequence setforth in SEQ ID NO: 2 or 3, and, optionally, a CDR3 having an amino acidsequence set forth in SEQ ID NO: 4; and 2) an immunoglobulin light chainvariable region comprising a CDR1 having an amino acid sequence setforth in SEQ ID NO: 16, a CDR2 having an amino acid sequence set forthin SEQ ID NO: 17, and, optionally, a CDR3 having an amino acid sequenceset forth in SEQ ID NO: 18. In certain embodiments, CDR2 of the heavychain variable region is SEQ ID NO: 2. In certain embodiments, CDR2 ofthe heavy chain variable region is SEQ ID NO: 3.

In certain embodiments, the anti-CD123/IL-3Rα antibodies andantigen-binding fragments thereof comprise: a) an immunoglobulin heavychain variable region comprising a CDR1 having an amino acid sequenceset forth in SEQ ID NO: 5, a CDR2 having an amino acid sequence setforth in SEQ ID NO: 6, 7, 8, 9, or 10, and, optionally, a CDR3 having anamino acid sequence set forth in SEQ ID NO: 11; and 2) an immunoglobulinlight chain variable region comprising a CDR1 having an amino acidsequence set forth in SEQ ID NO: 19 or 20, a CDR2 having an amino acidsequence set forth in SEQ ID NO: 21, and, optionally, a CDR3 having anamino acid sequence set forth in SEQ ID NO: 22. In certain embodiments,CDR2 of the heavy chain variable region is SEQ ID NO: 6, and CDR1 of thelight chain variable region is SEQ ID NO: 19. In certain embodiments,CDR2 of the heavy chain variable region is SEQ ID NO: 7, and CDR1 of thelight chain variable region is SEQ ID NO: 19. In certain embodiments,CDR2 of the heavy chain variable region is SEQ ID NO: 8, and CDR1 of thelight chain variable region is SEQ ID NO: 19. In certain embodiments,CDR2 of the heavy chain variable region is SEQ ID NO: 9, and CDR1 of thelight chain variable region is SEQ ID NO: 19. In certain embodiments,CDR2 of the heavy chain variable region is SEQ ID NO: 10, and CDR1 ofthe light chain variable region is SEQ ID NO: 19. In certainembodiments, CDR2 of the heavy chain variable region is SEQ ID NO: 6,and CDR1 of the light chain variable region is SEQ ID NO: 20. In certainembodiments, CDR2 of the heavy chain variable region is SEQ ID NO: 7,and CDR1 of the light chain variable region is SEQ ID NO: 20. In certainembodiments, CDR2 of the heavy chain variable region is SEQ ID NO: 8,and CDR1 of the light chain variable region is SEQ ID NO: 20. In certainembodiments, CDR2 of the heavy chain variable region is SEQ ID NO: 9,and CDR1 of the light chain variable region is SEQ ID NO: 20. In certainembodiments, CDR2 of the heavy chain variable region is SEQ ID NO: 10,and CDR1 of the light chain variable region is SEQ ID NO: 20. For eachpairwise combinations of heavy chain variable region CDR2 with lightchain variable region CDR1 above, the heavy chain variable region CDR1and 3 are SEQ ID NOs: 5 and 11, respectively, and the light chainvariable region CDR2 and 3 are SEQ ID NOs: 21 and 22, respectively.

In certain embodiments, the anti-CD123/IL-3Rα antibodies andantigen-binding fragments thereof comprise: a) an immunoglobulin heavychain variable region comprising a CDR1 having an amino acid sequenceset forth in SEQ ID NO: 12, a CDR2 having an amino acid sequence setforth in SEQ ID NO: 13 or 14, and, optionally, a CDR3 having an aminoacid sequence set forth in SEQ ID NO: 15; and 2) an immunoglobulin lightchain variable region comprising a CDR1 having an amino acid sequenceset forth in SEQ ID NO: 23, a CDR2 having an amino acid sequence setforth in SEQ ID NO: 24, and, optionally, a CDR3 having an amino acidsequence set forth in SEQ ID NO: 25. In certain embodiments, CDR2 of theheavy chain variable region is SEQ ID NO: 13. In certain embodiments,CDR2 of the heavy chain variable region is SEQ ID NO: 14.

In certain embodiments, CDR1 sequences from the light chain and heavychain of one antibody (such as SEQ ID NOs: 5 and 19) can be combinedwith CDR2 sequences from the light chain and heavy chain of anotherantibody (such as SEQ ID NOs: 2 and 17), and optionally can be combinedwith CDR3 sequences from the light chain and heavy chain of the same(e.g., SEQ ID NOs: 4 and 18, or 11 and 22) or yet another antibody(e.g., SEQ ID NOs: 15 and 25). All possible combinations based on SEQ IDNOs: 1-25 in Table 1, particularly those pertaining to the same antibodynumber (e.g., all six light chain and heavy chain CDRs come fromCD123-3, or from CD123-6, or from CD123-14) are contemplated hereinwithout exhaustively enumerating all the specific combinations.

In certain embodiments, the anti-CD123/IL-3Rα antibodies andantigen-binding fragments thereof have conserved amino acidsubstitutions over 1, 2, or 3 consecutive residues in any one or moreCDR sequences above. That is, in some embodiments, the subjectantibodies and antigen-binding fragments thereof may have conservedamino acid substitutions over 1, 2, or 3 consecutive residues, in anyone or more of SEQ ID NOs: 1-25.

In certain embodiments, the CD123/IL-3Rα-binding agents are CD123/IL-3Rαantibodies or antigen-binding fragments thereof that comprise a heavychain variable region (HCVR) and a light chain variable region (LCVR),wherein the HCVR and LCVR are any of the sequences provided in Tables 3Aand 4A below. Selected corresponding nucleic acid sequences encoding theHCVR and LCVR are in Tables 3B and 4B.

TABLE 3A Heavy Chain Variable Region Amino Acid Sequences AntibodyAlt Name VH Amino Acid Sequence (SEQ ID NO) CD123-3 CD123Mu-3EFQLQQSGPEVVKPGASVKMSCKASGYTFTSYVMHWMKQKPGQGLEWIGYIKPYKDGTKYNEKFKGKATLISDKPSSTAYMELSSLTSEDSAVYYCAREGENGYYDAMDYWGQGTSVTV SS (SEQ ID NO: 26) CD123-6CD123Mu-6 EFQLQQSGPELVKPGASVKMSCKASGYIFTSSIMHWMKQKPGQGLEWIGYIKPYNDGTKYNEKFKGKATLTSDKSSSTANMELNSLTSEDSAVYYCAREGGNDYYDTMDYWGQGTSVT VSS (SEQ ID NO: 28) CD123-14CD123Mu-14 EVKLVESGGDLVKPGGSLKLSCAASGFTFSNYAMSWVRQNSEKRLEWVATINSGGSFTYYPDSVKGRFTISRDNAKDSLYLQMSSLNSEDTAMYYCARQSEAYYGYDKRTWFAYWGQG TLVTVSS (SEQ ID NO: 30)huCD123-6Gvl QVQLVQSGAEVKKPGASVKVSCKASGYGFTSSIMHWVRQAPGQGLEWMGYIKPYNDGTKYNEKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAREGGNDYYDTMDYWGQGTL VTVSS (SEQ ID NO: 32)huCD123-6Gv6/7 QXQLVQSGAEVKKPGASVKVSCKASGYIFTSSIMHWVRQAPGQGLEWIGYIKPYNDGTKYNEKFKGRATLTSDRSTSTAYMELSSLRSEDTAVYYCAREGGNDYYDTMDYWGQGTLVT VSS (SEQ ID NO: 34) huCD123-6SXQLVQSGAEVKKPGASVKVSCKASGYIFTSSIMHWVRQA Gv6/7-NTS2PGQGLEWIGYIKPYNDGTKYNEKFKGRATLTSDRSTSTAYMELSSLRSEDTAVYYCAREGGNDYYDTMDYWGQGTLVT VSS (SEQ ID NO: 38)huCD123-6rhv1.0 QVQLVQSGAEVVKPGASVKMSCKASGYTFTSSIMHWMKQKPGQGLEWIGYIRPYNDGTRYNQKFQGKATLTSDRSSSTANMELNSLTSEDSAVYYCAREGGNDYYDTMDYWGQGTSV TVSS (SEQ ID NO: 39)huCD123-6rhv1.1 QFQLVQSGAEVVKPGASVKMSCKASGYTFTSSIMHWMKQKPGQGLEWIGYIKPYNDGTKYNQKFQGKATLTSDKSSSTANMELNSLTSEDSAVYYCAREGGNDYYDTMDYWGQGTSV TVSS (SEQ ID NO: 40) * In allsequences above in which the 2^(nd) residue from the N-terminus is X (orXaa), e.g., SEQ ID NOs: 34 and 38, X is F for Gv6 sequences, while X isV for Gv7 sequences.

TABLE 3B Selected Heavy Chain Variable Region Nucleic Acid SequencesAntibody VH DNA Sequence (SEQ ID NO) huCD123-6AAGCTTGCCACCATGGGATGGTCCTGCATTATCCTGTTCCT V_(H)GvlTGTAGCAACTGCAACAGGAGTCCACAGCCAGGTCCAACTGGTGCAGTCCGGGGCCGAGGTGAAGAAACCAGGCGCATCCGTGAAGGTCAGCTGTAAAGCCAGCGGCTATGGTTTTACCAGCTCAATCATGCACTGGGTCAGGCAAGCCCCAGGACAGGGTCTCGAATGGATGGGATACATTAAGCCTTACAATGATGGTACAAAATATAATGAAAAATTTAAGGGTCGTGTTACCATGACAAGGGATACATCAACTAGCACTGTCTATATGGAACTGAGCTCTCTCAGGTCCGAGGATACTGCAGTATATTACTGCGCCCGGGAGGGAGGCAACGACTATTACGACACCATGGACTATTGGGGGCAGGGCACACTGGTTACTGTATCCAGCGCCTCTACTA AGGGCCC (SEQ ID NO: 62)huCD123-6 AAGCTTGCCACCATGGGCTGGTCCTGTATCATCCTGTTCCT V_(H)Gv6CGTTGCAACAGCAACTGGCGTGCACAGCCAGTTCCAGCTTGTGCAGAGTGGCGCCGAAGTCAAGAAACCAGGCGCTAGTGTCAAGGTGTCCTGTAAGGCATCAGGCTACATCTTTACCAGCTCCATCATGCATTGGGTCAGACAGGCTCCTGGACAGGGCCTGGAGTGGATTGGGTATATCAAGCCATACAATGATGGGACAAAATACAATGAAAAGTTTAAAGGGCGAGCCACTCTGACATCTGATCGGAGTACAAGCACTGCCTACATGGAATTGAGCTCACTGCGGTCCGAAGACACTGCTGTGTATTATTGCGCTCGGGAGGGAGGGAACGACTACTACGATACCATGGACTACTGGGGCCAGGGCACCCTGGTTACCGTCAGCAGCGCTTCCACTAA GGGCCC (SEQ ID NO: 64)huCD123-6 AAGCTTGCCACCATGGGCTGGTCCTGTATCATCCTGTTCCT V_(H)Gv7CGTTGCAACAGCAACTGGCGTGCACAGCCAGGTCCAACTTGTGCAGAGTGGCGCCGAAGTCAAGAAACCAGGCGCTAGTGTCAAGGTGTCCTGTAAGGCATCAGGCTACATCTTTACCAGCTCCATCATGCATTGGGTCAGACAGGCTCCTGGACAGGGCCTGGAGTGGATTGGGTATATCAAGCCATACAATGATGGGACAAAATACAATGAAAAGTTTAAAGGGCGAGCCACTCTGACATCTGATCGGAGTACAAGCACTGCCTACATGGAATTGAGCTCACTGCGGTCCGAAGACACTGCTGTGTATTATTGCGCTCGGGAGGGAGGGAACGACTACTACGATACCATGGACTACTGGGGCCAGGGCACCCTGGTTACCGTCAGCAGCGCTTCCACTAA GGGCCC (SEQ ID NO: 66)huCD123-6 AAGCTTGCCACCATGGGGTGGAGCTGCATTATTCTGTTCTT V_(H)rhv1.0GGTCGCCACCGCAACTGGCGTCCACTCTCAGGTCCAGCTCGTCCAGTCTGGGGCAGAAGTGGTCAAGCCCGGTGCATCTGTGAAAATGTCCTGCAAAGCTAGCGGGTATACATTCACATCTAGTATCATGCATTGGATGAAACAGAAGCCTGGCCAGGGTCTGGAGTGGATAGGATATATCAGGCCTTACAACGATGGCACTCGATACAACCAAAAGTTCCAGGGTAAAGCTACACTGACCTCAGACCGCTCAAGCAGTACAGCAAACATGGAACTGAACAGTCTTACCTCTGAGGACAGTGCCGTTTACTATTGCGCCAGGGAGGGTGGCAATGACTACTATGATACTATGGACTACTGGGGACAGGGTACCTCTGTAACAGTTTCAAGCGCCAGCACTAA GGGCCC (SEQ ID NO: 68)huCD123-6 AAGCTTGCCACCATGGGCTGGTCTTGTATTATTCTGTTTCT V_(H)rhv1.1GGTGGCCACCGCAACAGGCGTTCACAGTCAATTCCAGCTGGTCCAGTCCGGCGCCGAGGTTGTCAAACCTGGTGCCAGCGTAAAGATGTCTTGCAAAGCTAGCGGCTATACTTTCACTTCTTCAATTATGCACTGGATGAAGCAAAAGCCTGGACAGGGCCTGGAATGGATCGGCTACATTAAACCTTATAACGACGGCACAAAGTACAATCAGAAGTTCCAAGGAAAGGCAACCCTGACCTCAGACAAGTCTTCATCCACTGCCAACATGGAACTTAATAGTCTTACCTCTGAGGATTCCGCTGTCTATTATTGCGCTCGGGAGGGGGGGAACGACTATTACGACACCATGGACTACTGGGGACAGGGCACCAGTGTTACCGTGTCCAGCGCTAGCACCAAG GGCCC (SEQ ID NO: 69) * Thebolded bases mark the first codon of the mature variable region aminoacid sequence

TABLE 4A Light Chain Variable Region Amino Acid Sequences AntibodyAlt Name VL Amino Acid Sequence (SEQ ID NO) CD123-3DIQMTQSPSSLSASLGGKVTITCKASQDINKYIAWYQHKPGKGPRLLIHYTSTLQPGIPSRFSGSGSGRDYSFSISNLEPEDIATYYCLQYDNLLYTFGGGTKLELKR(SEQ ID NO: 27) CD123-6DIKMTQSPSSMYASLGERVTITCKASQDINSYLSWFQQKPGKSPKTLIYRVNRLVDGVPSRFSGSGSGQDYSLTISSLEYEDMGIYYCLQYDAFPYTFGGGTKLEIKR(SEQ ID NO: 29) CD123-14DILLTQSPAILSVSPGTRVSFSCRASQSVGTSIHWYQQRPNGFPRLLIKYASESISGIPSRFSGSGSGTDFTLNINSVESEDIADYYCQQSKSWPLTFGAGTKLELKR(SEQ ID NO: 31) huCD123-6Gv 1DIQMTQSPSSLSASVGDRVTITCRASQDINSYLAWFQQKPGKAPKSLIYRVNRLVSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQYDAFPYTFGQGTKVEIKR (SEQ ID NO: 33) huCD123-6Gv4DIQMTQSPSSLSASVGDRVTITCRASQDINSYLSWFQQKPGKAPKTLIYRVNRLVDGVPSRFSGSGSGNDYTLTISSLQPEDFATYYCLQYDAFPYTFGQGTKVEIKR (SEQ ID NO: 35) huCD123-6Gv4-SIQMTQSPSSLSASVGDRVTITCRASQDINSYLSWFQQKPG NTS3KAPKTLIYRVNRLVDGVPSRFSGSGSGNDYTLTISSLQPEDFATYYCLQYDAFPYTFGQGTKVEIKR (SEQ ID NO: 37) huCD123-6r1v1.0DIQMTQSPSSMSASVGERVTITCRASQDINSYLSWFQQKPGKSPKTLIYRVNRLVDGVPSRFSGSGSGQDYSLTISSLEPEDMGIYYCLQYDAFPYTFGQGTKLEIKR (SEQ ID NO: 41)

TABLE 4B  elected Light Chain Variable Region Nucleic Acid SequencesAntibody Alt Name VL DNA Sequence (SEQ ID NO) huCD123-6GAATTCGCCACCATGGGTTGGTCTTGTATAATCCTGTTCC V_(L)Gv1TGGTCGCTACCGCAACAGGGGTTCACTCAGACATCCAGATGACCCAGAGTCCCTCTTCTCTGAGCGCTTCTGTTGGGGACCGGGTGACCATCACCTGTCGGGCATCCCAGGACATCAATTCTTACCTGGCTTGGTTCCAGCAGAAGCCCGGAAAAGCCCCTAAATCTCTCATTTACCGGGTAAACCGTTTGGTCTCCGGAGTGCCTTCAAGGTTTAGTGGATCTGGATCAGGTACAGACTTCACTCTCACCATAAGCAGCCTGCAACCAGAGGATTTCGCAACTTACTACTGCTTGCAGTATGACGCCTTCCCTTACACTTTCGGGCAGGGGACCAAAGTGGAAATAAAGCG TACG (SEQ ID NO: 63) huCD123-6GAATTCGCCACCATGGGTTGGTCCTGTATCATCCTCTTTC V_(L)Gv4TGGTGGCAACTGCAACCGGCGTCCATAGCGACATTCAGATGACACAGTCTCCTTCTTCCCTGAGCGCCAGCGTCGGGGACCGCGTGACTATCACATGTCGGGCCTCCCAGGACATTAACTCTTACCTCTCCTGGTTCCAGCAGAAGCCTGGGAAAGCCCCAAAGACACTGATATACAGGGTAAATCGTTTGGTTGACGGTGTACCATCACGATTTTCCGGTAGTGGGTCTGGAAACGATTACACTCTCACAATTAGCAGCCTGCAACCAGAGGACTTTGCAACATACTATTGCCTGCAGTACGATGCTTTTCCTTATACCTTCGGTCAGGGTACCAAGGTGGAAATTAAAC GTACG (SEQ ID NO: 65)huCD123-6V_(L) GAATTCGCCACCATGGGCTGGTCATGTATTATCCTGTTTC (resurfaced)TGGTTGCAACCGCAACAGGAGTACACTCTGATATCCAGATGACTCAGTCTCCCTCTTCTATGTCTGCTTCTGTGGGAGAGAGAGTCACCATCACCTGTCGCGCTTCCCAAGATATTAATAGCTATCTGTCTTGGTTCCAACAGAAACCTGGCAAATCACCCAAGACTCTGATTTATCGGGTTAACCGCCTGGTGGACGGTGTGCCTTCACGCTTCTCCGGCAGCGGTAGTGGACAAGACTATAGCCTGACAATTTCTTCTCTTGAACCCGAGGACATGGGAATCTACTATTGCTTGCAGTATGACGCTTTTCCTTATACATTCGGCCAGGGCACAAAGCTGGAAATCAAACG TACG (SEQ ID NO: 67) * The boldedbases mark the first codon of the mature variable region amino acidsequence.

In certain embodiments, the anti-CD123/IL-3Rα antibodies andantigen-binding fragments thereof comprises: (a) a V_(H) sequence atleast 95% identical to a reference V_(H) sequence selected from a grouphaving amino acid sequences represented by SEQ ID NOs: 26, 28, 30, 32,34, 38, 39, and 40 (or SEQ ID NOs: 26, 28, 30, 32, 34, and 38); and/or(b) a V_(L) sequence at least 95% identical to a reference V_(L)sequence selected from the group having amino acid sequences representedby SEQ ID NOs: 27, 29, 31, 33, 35, 37, and 41 (or SEQ ID NOs: 27, 29,31, 35, and 37).

In certain embodiments, the anti-CD123/IL-3Rα antibodies andantigen-binding fragments thereof comprises: (a) a V_(H) sequence atleast 96% identical to a reference V_(H) sequence selected from a grouphaving amino acid sequences represented by SEQ ID NOs: 26, 28, 30, 32,34, 38, 39, and 40 (or SEQ ID NOs: 26, 28, 30, 32, 34, and 38); and/or(b) a V_(L) sequence at least 96% identical to a reference V_(L)sequence selected from the group having amino acid sequences representedby SEQ ID NOs: 27, 29, 31, 33, 35, 37, and 41 (or SEQ ID NOs: 27, 29,31, 35, and 37).

In certain embodiments, the anti-CD123/IL-3Rα antibodies andantigen-binding fragments thereof comprises: (a) a V_(H) sequence atleast 97% identical to a reference V_(H) sequence selected from a grouphaving amino acid sequences represented by SEQ ID NOs: 26, 28, 30, 32,34, 38, 39, and 40 (or SEQ ID NOs: 26, 28, 30, 32, 34, and 38); and/or(b) a V_(L) sequence at least 97% identical to a reference V_(L)sequence selected from the group having amino acid sequences representedby SEQ ID NOs: 27, 29, 31, 33, 35, 37, and 41 (or SEQ ID NOs: 27, 29,31, 35, and 37).

In certain embodiments, the anti-CD123/IL-3Rα antibodies andantigen-binding fragments thereof comprises: (a) a V_(H) sequence atleast 98% identical to a reference V_(H) sequence selected from a grouphaving amino acid sequences represented by SEQ ID NOs: 26, 28, 30, 32,34, 38, 39, and 40 (or SEQ ID NOs: 26, 28, 30, 32, 34, and 38); and/or(b) a V_(L) sequence at least 98% identical to a reference V_(L)sequence selected from the group having amino acid sequences representedby SEQ ID NOs: 27, 29, 31, 33, 35, 37, and 41 (or SEQ ID NOs: 27, 29,31, 35, and 37).

In certain embodiments, the anti-CD123 antibodies and antigen-bindingfragments thereof comprises: (a) a V_(H) sequence at least 99% identicalto a reference V_(H) sequence selected from a group having amino acidsequences represented by SEQ ID NOs: 26, 28, 30, 32, 34, 38, 39, and 40(or SEQ ID NOs: 26, 28, 30, 32, 34, and 38); and/or (b) a V_(L) sequenceat least 99% identical to a reference V_(L) sequence selected from thegroup having amino acid sequences represented by SEQ ID NOs: 27, 29, 31,33, 35, 37, and 41 (or SEQ ID NOs: 27, 29, 31, 35, and 37).

In certain embodiments, the CD123/IL-3Rα antibody/antigen-bindingfragment thereof having a certain percentage of sequence identity to SEQID NOs: 26, 28, 30, 32, 34, 38, 39, and 40 (preferably SEQ ID NOs: 26,28, 30, 32, 34, and 38) and/or 27, 29, 31, 33, 35, 37, and 41 (or SEQ IDNOs: 27, 29, 31, 35, and 37) differs from SEQ ID NOs: 26, 28, 30, 32,34, 38, 39, and 40 (or SEQ ID NOs: 26, 28, 30, 32, 34, and 38) and/or27, 29, 31, 33, 35, 37, and 41 (or SEQ ID NOs: 27, 29, 31, 35, and 37)by conservative amino acid substitutions only, such as 1, 2, or 3conservative amino acid substitutions. In certain embodiments, theconservative amino acid substitutions are substitutions of 1, 2, or 3consecutive amino acids in one or more CDR regions of the heavy and/orlight chains.

In certain embodiments, the anti-CD123/IL-3Rα antibodies andantigen-binding fragments thereof comprises: (a) a V_(H) sequenceidentical to a reference V_(H) sequence selected from a group havingamino acid sequences represented by SEQ ID NOs: 26, 28, 30, 32, 34, 38,39, and 40 (or SEQ ID NOs: 26, 28, 30, 32, 34, and 38); and/or (b) aV_(L) sequence identical to a reference V_(L) sequence selected from thegroup having amino acid sequences represented by SEQ ID NOs: 27, 29, 31,33, 35, 37, and 41 (or SEQ ID NOs: 27, 29, 31, 35, and 37).

In certain embodiments, the anti-CD123/IL-3Rα antibodies andantigen-binding fragments thereof comprises: a V_(H) sequence as setforth in SEQ ID NO: 26, and/or a V_(L) sequence as set forth in SEQ IDNO: 27.

In certain embodiments, the anti-CD123/IL-3Rα antibodies andantigen-binding fragments thereof comprises: a V_(H) sequence as setforth in SEQ ID NO: 28, and/or a V_(L) sequence as set forth in SEQ IDNO: 29.

In certain embodiments, the anti-CD123/IL-3Rα antibodies andantigen-binding fragments thereof comprises: a V_(H) sequence as setforth in SEQ ID NO: 30, and/or a V_(L) sequence as set forth in SEQ IDNO: 31.

In certain embodiments, the anti-CD123/IL-3Rα antibodies andantigen-binding fragments thereof comprises: a V_(H) sequence as setforth in SEQ ID NO: 34, and/or a V_(L) sequence as set forth in SEQ IDNO: 35.

In certain embodiments, the anti-CD123/IL-3Rα antibodies andantigen-binding fragments thereof comprises a V_(H) sequence and a V_(L)sequence with a combination of SEQ ID NOs. selected from the groupconsisting of: 32/33, 34/33, 38/33, 39/33, 40/33, 32/35, 34/35, 38/35,39/35, 40/35, 32/37, 34/37, 38/37, 39/37, 40/37, 39/33, 39/35, 39/37,39/41, 40/33, 40/35, 40/37, and 40/41.

For example, in one embodiment, the anti-CD123/IL-3Rα antibodies andantigen-binding fragments thereof comprises: a) an immunoglobulin heavychain variable region having the amino acid sequence set forth in SEQ IDNO: 39 or 40; and, b) an immunoglobulin light chain variable regionhaving the amino acid sequence set forth in SEQ ID NO: 41. In certainembodiments, the V_(H) sequence is set forth in SEQ ID NO: 39, and theV_(L) sequence is set forth in SEQ ID NO: 41. In certain embodiments,the V_(H) sequence is set forth in SEQ ID NO: 40, and the V_(L) sequenceis set forth in SEQ ID NO: 41.

In a related embodiment, the anti-CD123/IL-3Rα antibodies andantigen-binding fragments thereof comprises: a) an immunoglobulin heavychain variable region having the amino acid sequence set forth in SEQ IDNO: 34; and, b) an immunoglobulin light chain variable region having theamino acid sequence set forth in SEQ ID NO: 35. In certain embodiments,Xaa in SEQ ID NO: 34 is Phe (F). In certain embodiments, Xaa in SEQ IDNO: 34 is Val (V).

In another embodiment, the anti-CD123/IL-3Rα antibodies andantigen-binding fragments thereof comprises: a) an immunoglobulin heavychain variable region having the amino acid sequence set forth in SEQ IDNO: 39 or 40, except that the first residue is replaced by Ser (S); and,b) an immunoglobulin light chain variable region having the amino acidsequence set forth in SEQ ID NO: 41.

In another embodiment, the anti-CD123/IL-3Rα antibodies andantigen-binding fragments thereof comprises: a) an immunoglobulin heavychain variable region having the amino acid sequence set forth in SEQ IDNO: 39 or 40; and, b) an immunoglobulin light chain variable regionhaving the amino acid sequence set forth in SEQ ID NO: 41, except thatthe first residue is replaced by Ser (S).

In another embodiment, the anti-CD123/IL-3Rα antibodies andantigen-binding fragments thereof comprises: a) an immunoglobulin heavychain region having the amino acid sequence set forth in SEQ ID NO: 59or 60, except that the N-terminal residue is Ser, and except that theresidue corresponding to the 5^(th) to the last residue of SEQ ID NO: 54is Cys; and b) an immunoglobulin light chain variable region having theamino acid sequence set forth in SEQ ID NO: 41.

In another embodiment, the anti-CD123/IL-3Rα antibodies andantigen-binding fragments thereof comprises: a) an immunoglobulin heavychain region having the amino acid sequence set forth in SEQ ID NO: 59or 60, except that the residue corresponding to the 5^(th) to the lastresidue of SEQ ID NO: 54 is Cys; and b) an immunoglobulin light chainvariable region having the amino acid sequence set forth in SEQ ID NO:41, except that the N-terminal residue is Ser.

In another embodiment, the anti-CD123/IL-3Rα antibodies andantigen-binding fragments thereof comprises: a) an immunoglobulin heavychain variable region having the amino acid sequence set forth in SEQ IDNO: 38; and, b) an immunoglobulin light chain variable region having theamino acid sequence set forth in SEQ ID NO: 35. In certain embodiments,Xaa in SEQ ID NO: 38 is Phe (F). In certain embodiments, Xaa in SEQ IDNO: 38 is Val (V).

In another embodiment, the anti-CD123/IL-3Rα antibodies andantigen-binding fragments thereof comprises: a) an immunoglobulin heavychain variable region having the amino acid sequence set forth in SEQ IDNO: 34; and, b) an immunoglobulin light chain variable region having theamino acid sequence set forth in SEQ ID NO: 37. In certain embodiments,Xaa in SEQ ID NO: 34 is Phe (F). In certain embodiments, Xaa in SEQ IDNO: 34 is Val (V).

In another embodiment, the anti-CD123/IL-3Rα antibodies andantigen-binding fragments thereof comprises: a) an immunoglobulin heavychain region having the amino acid sequence set forth in SEQ ID NO: 56;and, b) an immunoglobulin light chain variable region having the aminoacid sequence set forth in SEQ ID NO: 35. In certain embodiments, Xaa inSEQ ID NO: 56 is Phe (F). In certain embodiments, Xaa in SEQ ID NO: 56is Val (V).

In another embodiment, the anti-CD123/IL-3Rα antibodies andantigen-binding fragments thereof comprises: a) an immunoglobulin heavychain region having the amino acid sequence set forth in SEQ ID NO: 54;and, b) an immunoglobulin light chain variable region having the aminoacid sequence set forth in SEQ ID NO: 37. In certain embodiments, Xaa inSEQ ID NO: 54 is Phe (F). In certain embodiments, Xaa in SEQ ID NO: 54is Val (V).

In another embodiment, the anti-CD123/IL-3Rα antibodies andantigen-binding fragments thereof comprises: a) an immunoglobulin heavychain region having the amino acid sequence set forth in SEQ ID NO: 59or 60, except that the residue corresponding to the 5^(th) to the lastresidue of SEQ ID NO: 54 is Cys; and b) an immunoglobulin light chainvariable region having the amino acid sequence set forth in SEQ ID NO:41.

In another embodiment, the anti-CD123/IL-3Rα antibodies andantigen-binding fragments thereof comprises: a) an immunoglobulin heavychain region having the amino acid sequence set forth in SEQ ID NO: 54;and, b) an immunoglobulin light chain variable region having the aminoacid sequence set forth in SEQ ID NO: 35. In certain embodiments, Xaa inSEQ ID NO: 54 is Phe (F). In certain embodiments, Xaa in SEQ ID NO: 54is Val (V).

In another embodiment, the anti-CD123/IL-3Rα antibodies andantigen-binding fragments thereof comprises: a) an immunoglobulin heavychain region having the amino acid sequence set forth in SEQ ID NO: 56;and, b) an immunoglobulin light chain variable region having the aminoacid sequence set forth in SEQ ID NO: 35. In certain embodiments, Xaa inSEQ ID NO: 56 is Phe (F). In certain embodiments, Xaa in SEQ ID NO: 56is Val (V).

In another embodiment, the anti-CD123/IL-3Rα antibodies andantigen-binding fragments thereof comprises: a) an immunoglobulin heavychain region having the amino acid sequence set forth in SEQ ID NO: 54;and, b) an immunoglobulin light chain variable region having the aminoacid sequence set forth in SEQ ID NO: 37. In certain embodiments, Xaa inSEQ ID NO: 54 is Phe (F). In certain embodiments, Xaa in SEQ ID NO: 54is Val (V).

In certain embodiments, the anti-CD123/IL-3Rα antibodies andantigen-binding fragments thereof specifically binds CD123/IL-3Rα. Incertain embodiments, the CD123/IL-3Rα antibody or antigen-bindingfragment thereof is a murine, chimeric, humanized, or human antibody orantigen-binding fragment thereof that specifically binds CD123/IL-3Rα.In certain embodiments, the humanized antibody or antigen-bindingfragment thereof is a CDR-grafted or resurfaced antibody orantigen-binding fragment thereof.

In certain embodiments, the anti-CD123/IL-3Rα antibodies are full-lengthantibodies. The full-length antibodies may comprise any of theantibodies above defined by the 1-4 CDR (e.g., CDR1 and CDR2 of theheavy chain; CDR1 and CDR2 of the heavy and light chains), 1-6 CDRsequences (e.g., CDR1-CDR3 of the heavy chain; CDR1-CDR3 of the heavyand light chains), or any of the antibodies above defined by the LCVRand/or the HCVR, or any of the full-length antibodies having a heavychain sequence in Table 5, or any of the full-length antibodies having alight chain sequence in Table 6, or any of the full-length antibodieshaving a heavy chain sequence in Table 5 and a light chain sequence inTable 6.

TABLE 5 Full-Length Heavy Chain Amino Acid Sequences AntibodyFull-Length Heavy Chain Amino Acid Sequence (SEQ ID NO) CD123-3EFQLQQSGPEVVKPGASVKMSCKASGYTFTSYVMHWMKQKPGQGLEWIGYIKPYKDGTKYNEKFKGKATLISDKPSSTAYMELSSLTSEDSAVYYCAREGENGYYDAMDYWGQGTSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLESDLYTLSSSVTVPSSPRPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMNTNGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK (SEQ ID NO: 42) CD123-6EFQLQQSGPELVKPGASVKMSCKASGYIFTSSIMHWMKQKPGQGLEWIGYIKPYNDGTKYNEKFKGKATLTSDKSSSTANMELNSLTSEDSAVYYCAREGGNDYYDTMDYWGQGTSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLESDLYTLSSSVTVPSSMRPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMNTNGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK (SEQ ID NO: 44) CD123-14EVKLVESGGDLVKPGGSLKLSCAASGFTFSNYAMSWVRQNSEKRLEWVATINSGGSFTYYPDSVKGRFTISRDNAKDSLYLQMSSLNSEDTAMYYCARQSEAYYGYDKRTWFAYWGQGTLVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLESDLYTLSSSVTVPSSPRPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMNTNGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK (SEQ ID NO: 46) huCD123-QVQLVQSGAEVKKPGASVKVSCKASGYGFTSSIMHWVRQAPGQGLEWMGY 6Gv1IKPYNDGTKYNEKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAREGGNDYYDTMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 48) huCD123-QXQLVQSGAEVKKPGASVKVSCKASGYIFTSSIMHWVRQAPGQGLEWIGYIK 6Gv6/7PYNDGTKYNEKFKGRATLTSDRSTSTAYMELSSLRSEDTAVYYCAREGGNDYYDTMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 50) huCD123-SXQLVQSGAEVKKPGASVKVSCKASGYIFTSSIMHWVRQAPGQGLEWIGYIK 6 Gv6/7-PYNDGTKYNEKFKGRATLTSDRSTSTAYMELSSLRSEDTAVYYCAREGGND NTS2YYDTMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFP (or “S2”)EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 53) huCD123-QXQLVQSGAEVKKPGASVKVSCKASGYIFTSSIMHWVRQAPGQGLEWIGYIK 6 Gv6/7-PYNDGTKYNEKFKGRATLTSDRSTSTAYMELSSLRSEDTAVYYCAREGGND CysMabYYDTMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPG (SEQ ID NO: 54) huCD123-SXQLVQSGAEVKKPGASVKVSCKASGYIFTSSIMHWVRQAPGQGLEWIGYIK 6 Gv6/752-PYNDGTKYNEKFKGRATLTSDRSTSTAYMELSSLRSEDTAVYYCAREGGND CysMabYYDTMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPG (SEQ ID NO: 56) huCD123-QVQLVQSGAEVVKPGASVKMSCKASGYTFTSSIMHWMKQKPGQGLEWIGYI 6rhv1.0RPYNDGTRYNQKFQGKATLTSDRSSSTANMELNSLTSEDSAVYYCAREGGNDYYDTMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 59) huCD123-QFQLVQSGAEVVKPGASVKMSCKASGYTFTSSIMHWMKQKPGQGLEWIGYI 6rhv1.1KPYNDGTKYNQKFQGKATLTSDKSSSTANMELNSLTSEDSAVYYCAREGGNDYYDTMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 60) * In all sequencesabove in which the 2^(nd) residue from the N-terminus is X (or Xaa),e.g., SEQ ID NOs: 50, 53, 54, and 56, X is F for Gv6 sequences, while Xis V for Gv7 sequences. In some embodiments, the Met (bolded) in SEQ IDNO: 44 is Pro.

TABLE 6 Full-Length Light Chain Amino Acid Sequences AntibodyFull-length Light Chain Amino Acid Sequence (SEQ ID NO) CD123-3DIQMTQSPSSLSASLGGKVTITCKASQDINKYIAWYQHKPGKGPRLLIHYTSTLQPGIPSRFSGSGSGRDYSFSISNLEPEDIATYYCLQYDNLLYTFGGGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC (SEQ ID NO: 43) CD123-6DIKMTQSPSSMYASLGERVTITCKASQDINSYLSWFQQKPGKSPKTLIYRVNRLVDGVPSRFSGSGSGQDYSLTISSLEYEDMGIYYCLQYDAFPYTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC (SEQ ID NO: 45) CD123-14DILLTQSPAILSVSPGTRVSFSCRASQSVGTSIHWYQQRPNGFPRLLIKYASESISGIPSRFSGSGSGTDFTLNINSVESEDIADYYCQQSKSWPLTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC (SEQ ID NO: 47) huCD123-DIQMTQSPSSLSASVGDRVTITCRASQDINSYLAWFQQKPGKAPKSLIYRVN 6Gv1RLVSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQYDAFPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 49) huCD123-DIQMTQSPSSLSASVGDRVTITCRASQDINSYLSWFQQKPGKAPKTLIYRVN 6Gv4RLVDGVPSRFSGSGSGNDYTLTISSLQPEDFATYYCLQYDAFPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 51) huCD123-SIQMTQSPSSLSASVGDRVTITCRASQDINSYLSWFQQKPGKAPKTLIYRVN 6Gv4-RLVDGVPSRFSGSGSGNDYTLTISSLQPEDFATYYCLQYDAFPYTFGQGTK NTS3 (orVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNAL “S3”)QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 58) huCD123-DIQMTQSPSSMSASVGERVTITCRASQDINSYLSWFQQKPGKSPKTLIYRVN 6r1v1.0RLVDGVPSRFSGSGSGQDYSLTISSLEPEDMGIYYCLQYDAFPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 61)

In certain embodiments, the anti-CD123/IL-3Rα antibodies are full-lengthantibodies comprising: (a) a heavy chain having at least about 90%, 95%,96%, 97%, 98%, or 99% sequence identity to any of the full-length heavychain sequences above, such as any of the full-length heavy chainsequences in Table 5; and/or (b) a light chain having at least about90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of thefull-length light chain sequences above, such as any of the full-lengthlight chain sequences in Table 6. In certain embodiments, theanti-CD123/IL-3Rα antibodies are full-length antibodies comprising afull-length heavy chain sequence and a full-length light chain sequencecombination selected from the group consisting of SEQ ID NOs: 42/43,44/45, 46/47, 48/49, 50/49, 53/49, 54/49, 56/49, 59/49, 60/49, 48/51,50/51, 53/51, 54/51, 56/51, 59/51, 60/51, 48/58, 50/58, 53/58, 54/58,56/58, 59/58, 60/58, 59/49, 59/51, 59/58, 59/61, 60/49, 60/51, 60/58,and 60/61, or antibodies with at least about 90%, 95%, 96%, 97%, 98%, or99% sequence identity to any of the full-length heavy chain sequencesand/or light chain sequences thereof.

In certain embodiments, the anti-CD123/IL-3Rα antibodies are full-lengthantibodies comprising a full-length heavy chain sequence and afull-length light chain sequence combination selected from the groupconsisting of SEQ ID NOs: 59/61, and 60/61, or antibodies with at leastabout 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of thefull-length heavy chain sequences and/or light chain sequences thereof.Such antibodies may further comprise engineered N-terminal Ser/Thr inthe light chain, heavy chain, or both. Such antibodies may furthercomprise engineered Cys in the heavy chain CH3 domain in a positioncorresponding to the 5^(th) to the last Cys of SEQ ID NO: 54.

In certain embodiments, the anti-CD123/IL-3Rα antibody is a murine,chimeric, humanized, or human antibody that specifically bindsCD123/IL-3Rα. In certain embodiments, the anti-CD123/IL-3Rα antibodyhaving a certain percentage of sequence identity to any of thefull-length SEQ ID NOs differs from such SEQ ID NOs by conservativeamino acid substitutions only, e.g., by 1, 2, 3, 4, or 5 consecutiveconservative amino acid substitutions only. In certain embodiments, theconservative amino acid substitutions are outside the CDRs.

In certain embodiments, the antigen-binding fragment thereof is orcomprises a Fab, Fd, Fab′, F(ab′)₂, single chain Fv or scFv, disulfidelinked Fv, V-NAR domain, IgNar, intrabody, IgGΔCH2, minibody, F(ab′)₃,tetrabody, triabody, diabody, single-domain antibody, DVD-Ig, Fcab,mAb2, (scFv)₂, or scFv-Fc, of any one of the above antibodies.

In a related aspect, the invention also provides a polypeptidecomprising any of the antibodies or antigen-binding fragments thereof,any of the V_(H) and/or V_(L) sequences above, any of the HCVR and/orLCVR above, or any of the CDR sequence(s) of the HCVR and/or LCVR above.The polypeptide maybe, for example, a fusion with a non-antibody proteinor domain. In certain embodiments, the fusion protein is not a fusionwith a pseudomonas toxin.

The affinity or avidity of an antibody for an antigen can be determinedexperimentally using any suitable method well known in the art, e.g.,flow cytometry, enzyme-linked immunoabsorbent assay (ELISA), orradioimmunoassay (RIA), or kinetics (e.g., BIACORE™ analysis). Directbinding assays as well as competitive binding assay formats can bereadily employed. See, for example, Berzofsky et al., “Antibody-AntigenInteractions,” in Fundamental Immunology, Paul, W. E., Ed., Raven Press:New York, N.Y. (1984); Kuby, Janis Immunology, W. H. Freeman andCompany: New York, N.Y. (1992); and methods described herein.

The measured affinity of a particular antibody-antigen interaction canvary if measured under different conditions (e.g., salt concentration,pH, temperature). Thus, measurements of affinity and otherantigen-binding parameters (e.g., K_(D) or K_(d), k_(on), k_(off)) aremade with standardized solutions of antibody and antigen, and astandardized buffer, as known in the art and such as the bufferdescribed herein.

In one aspect, binding assays can be performed using flow cytometry oncells expressing the CD123/IL-3Rα antigen on the surface. For example,CD123/IL-3Rα-positive cells can be incubated with varying concentrationsof anti-CD123/IL-3Rα antibodies using 1×10⁵ cells per sample in 100 μLFACS buffer (e.g., RPMI-1640 medium supplemented with 2% normal goatserum). Then, the cells can be pelleted, washed, and incubated for 1 hrwith 100 μL of FITC-conjugated goat-anti-mouse or goat-anti-humanIgG-antibody (such as is obtainable from, for example JacksonLaboratory, 6 μg/mL in FACS buffer). The cells are then pelleted again,washed with FACS buffer and resuspended in 200 μL of PBS containing 1%formaldehyde. Samples can be acquired, for example, using a FACSCaliburflow cytometer with the HTS multiwell sampler and analyzed usingCellQuest Pro (all from BD Biosciences, San Diego, US). For each samplethe mean fluorescence intensity for FL1 (MFI) can be exported andplotted against the antibody concentration in a semi-log plot togenerate a binding curve. A sigmoidal dose-response curve is fitted forbinding curves and EC₅₀ values are calculated using programs such asGraphPad Prism v4 with default parameters (GraphPad software, San Diego,Calif.). EC₅₀ values can be used as a measure for the apparentdissociation constant “K_(d)” or “K_(D)” for each antibody.

Monoclonal antibodies can be prepared using hybridoma methods, such asthose described by Kohler and Milstein (1975) Nature 256:495. Using thehybridoma method, a mouse, hamster, or other appropriate host animal, isimmunized to elicit the production by lymphocytes of antibodies thatwill specifically bind to an immunizing antigen. Lymphocytes can also beimmunized in vitro. Following immunization, the lymphocytes are isolatedand fused with a suitable myeloma cell line using, for example,polyethylene glycol, to form hybridoma cells that can then be selectedaway from unfused lymphocytes and myeloma cells. Hybridomas that producemonoclonal antibodies directed specifically against a chosen antigen asdetermined by immunoprecipitation, immunoblotting, or by an in vitrobinding assay (e.g., radioimmunoassay (RIA); enzyme-linked immunosorbentassay (ELISA)) can then be propagated either in vitro culture usingstandard methods (Goding, Monoclonal Antibodies: Principles andPractice, Academic Press, 1986) or in vivo as ascites tumors in ananimal. The monoclonal antibodies can then be purified from the culturemedium or ascites fluid as described for polyclonal antibodies.

Alternatively monoclonal antibodies can also be made using recombinantDNA methods as described in U.S. Pat. No. 4,816,567. The polynucleotidesencoding a monoclonal antibody are isolated from mature B-cells orhybridoma cells, such as by RT-PCR using oligonucleotide primers thatspecifically amplify the genes encoding the heavy and light chains ofthe antibody, and their sequence is determined using conventionalprocedures. The isolated polynucleotides encoding the heavy and lightchains are then cloned into suitable expression vectors, which whentransfected into host cells such as E. coli cells, simian COS cells,Chinese hamster ovary (CHO) cells, or myeloma cells that do nototherwise produce immunoglobulin protein, monoclonal antibodies aregenerated by the host cells. Also, recombinant monoclonal antibodies orfragments thereof of the desired species can be isolated from phagedisplay libraries expressing CDRs of the desired species as described(McCafferty et al., Nature 348:552-554, 1990; Clackson et al., Nature,352:624-628, 1991; and Marks et al., J. Mol. Biol. 222:581-597, 1991).

The polynucleotide(s) encoding a monoclonal antibody can further bemodified in a number of different manners using recombinant DNAtechnology to generate alternative antibodies. In some embodiments, theconstant domains of the light and heavy chains of, for example, a mousemonoclonal antibody can be substituted 1) for those regions of, forexample, a human antibody to generate a chimeric antibody, or, 2) for anon-immunoglobulin polypeptide to generate a fusion antibody. In someembodiments, the constant regions are truncated or removed to generatethe desired antibody fragment of a monoclonal antibody. Site-directed orhigh-density mutagenesis of the variable region can be used to optimizespecificity, affinity, etc. of a monoclonal antibody.

In some embodiments, the monoclonal antibody against the humanCD123/IL-3Rα is a humanized antibody. In certain embodiments, suchantibodies are used therapeutically to reduce antigenicity and HAMA(human anti-mouse antibody) responses when administered to a humansubject.

Methods for engineering, humanizing or resurfacing non-human or humanantibodies can also be used and are well known in the art. A humanized,resurfaced or similarly engineered antibody can have one or more aminoacid residues from a source that is non-human, e.g., but not limited to,mouse, rat, rabbit, non-human primate or other mammal. These non-humanamino acid residues are replaced by residues that are often referred toas “import” residues, which are typically taken from an “import”variable, constant or other domain of a known human sequence.

Such imported sequences can be used to reduce immunogenicity or reduce,enhance or modify binding, affinity, on-rate, off-rate, avidity,specificity, half-life, or any other suitable characteristic, as knownin the art. In general, the CDR residues are directly and mostsubstantially involved in influencing CD123/IL-3Rα binding. Accordingly,part or all of the non-human or human CDR sequences are maintained whilethe non-human sequences of the variable and constant regions can bereplaced with human or other amino acids.

Antibodies can also optionally be humanized, resurfaced, engineered orhuman antibodies engineered with retention of high affinity for theantigen CD123/IL-3Rα and other favorable biological properties. Toachieve this goal, humanized (or human) or engineered anti-CD123/IL-3Rαantibodies and resurfaced antibodies can be optionally prepared by aprocess of analysis of the parental sequences and various conceptualhumanized and engineered products using three-dimensional models of theparental, engineered, and humanized sequences. Three-dimensionalimmunoglobulin models are commonly available and are familiar to thoseskilled in the art. Computer programs are available which illustrate anddisplay probable three-dimensional conformational structures of selectedcandidate immunoglobulin sequences. Inspection of these displays permitsanalysis of the likely role of the residues in the functioning of thecandidate immunoglobulin sequence, i.e., the analysis of residues thatinfluence the ability of the candidate immunoglobulin to bind itsantigen, such as CD123/IL-3Rα. In this way, framework (FR) residues canbe selected and combined from the consensus and import sequences so thatthe desired antibody characteristic, such as increased affinity for thetarget antigen(s), is achieved.

Humanization, resurfacing or engineering of antibodies of the presentinvention can be performed using any known method, such as but notlimited to those described in, Winter (Jones et al., Nature 321:522,1986; Riechmann et al., Nature 332:323, 1988; Verhoeyen et al., Science239:1534, 1988, Sims et al., J. Immunol. 151:2296, 1993; Chothia andLesk, J. Mol. Biol. 196:901, 1987, Carter et al., Proc. Natl. Acad. Sci.U.S.A. 89:4285, 1992; Presta et al., J. Immunol. 151:2623, 1993; Raguskaet al., Proc. Natl. Acad. Sci. U.S.A. 91(3):969-973, 1994; U.S. Pat.Nos. 5,639,641, 5,723,323; 5,976,862; 5,824,514; 5,817,483; 5,814,476;5,763,192; 5,723,323; 5,766,886; 5,714,352; 6,204,023; 6,180,370;5,693,762; 5,530,101; 5,585,089; 5,225,539; 4,816,567; PCT/: US98/16280;US96/18978; US91/09630; US91/05939; US94/01234; GB89/01334; GB91/01134;GB92/01755; WO90/14443; WO90/14424; WO90/14430; EP 229246; U.S. Pat.Nos. 7,557,189; 7,538,195; and 7,342,110, each of which is entirelyincorporated herein by reference, including the references citedtherein.

In certain alternative embodiments, the antibody to CD123/IL-3Rα is ahuman antibody. Human antibodies can be directly prepared using varioustechniques known in the art. Immortalized human B lymphocytes immunizedin vitro or isolated from an immunized individual that produce anantibody directed against a target antigen can be generated (See, e.g.,Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p.77 (1985); Boemer et al., 1991, J. Immunol, 147 (1):86-95; and U.S. Pat.No. 5,750,373). Also, the human antibody can be selected from a phagelibrary, where that phage library expresses human antibodies, asdescribed, for example, in Vaughan et al., Nat. Biotech. 14:309-314,1996, Sheets et al., Proc. Nat'l. Acad. Sci. 95:6157-6162, 1998,Hoogenboom and Winter, J. Mol. Biol. 227:381, 1991, and Marks et al., J.Mol. Biol. 222:581, 1991). Techniques for the generation and use ofantibody phage libraries are also described in U.S. Pat. Nos. 5,969,108,6,172,197, 5,885,793, 6,521,404; 6,544,731; 6,555,313; 6,582,915;6,593,081; 6,300,064; 6,653,068; 6,706,484; and 7,264,963; and Rothe etal., J. Mol. Bio. doi: 10.1016/j.jmb.2007.12.018, 2007 (each of which isincorporated by reference in its entirety). Affinity maturationstrategies and chain shuffling strategies (Marks et al., Bio/Technology10:779-783, 1992, incorporated by reference in its entirety) are knownin the art and can be employed to generate high affinity humanantibodies.

Humanized antibodies can also be made in transgenic mice containinghuman immunoglobulin loci that are capable upon immunization ofproducing the full repertoire of human antibodies in the absence ofendogenous immunoglobulin production. This approach is described in U.S.Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and5,661,016.

In certain embodiments are provided an antibody fragment to, forexample, increase tumor penetration. Various techniques are known forthe production of antibody fragments. Traditionally, these fragments arederived via proteolytic digestion of intact antibodies (for exampleMorimoto et al., Journal of Biochemical and Biophysical Methods 24:107-117, 1993; Brennan et al., Science 229:81, 1985). In certainembodiments, antibody fragments are produced recombinantly. Fab, Fv, andscFv antibody fragments can all be expressed in and secreted from E.coli or other host cells, thus allowing the production of large amountsof these fragments. Such antibody fragments can also be isolated fromantibody phage libraries. The antibody fragment can also be linearantibodies as described in U.S. Pat. No. 5,641,870, for example, and canbe monospecific or bispecific. Other techniques for the production ofantibody fragments will be apparent to the skilled practitioner.

For the purposes of the present invention, it should be appreciated thatmodified antibodies can comprise any type of variable region thatprovides for the association of the antibody with the polypeptides of ahuman CD123/IL-3Rα. In this regard, the variable region can comprise orbe derived from any type of mammal that can be induced to mount ahumoral response and generate immunoglobulins against the desired tumorassociated antigen. As such, the variable region of the modifiedantibodies can be, for example, of human, murine, non-human primate(e.g., cynomolgus monkeys, macaques, etc.) or lupine origin. In someembodiments both the variable and constant regions of the modifiedimmunoglobulins are human. In other embodiments the variable regions ofcompatible antibodies (usually derived from a non-human source) can beengineered or specifically tailored to improve the binding properties orreduce the immunogenicity of the molecule. In this respect, variableregions useful in the present invention can be humanized or otherwisealtered through the inclusion of imported amino acid sequences.

In certain embodiments, the variable domains in both the heavy and lightchains are altered by at least partial replacement of one or more CDRsand, if necessary, by partial framework region replacement and sequencechanging. Although the CDRs can be derived from an antibody of the sameclass or even subclass as the antibody from which the framework regionsare derived, it is envisaged that the CDRs will be derived from anantibody of different class and in certain embodiments from an antibodyfrom a different species. It may not be necessary to replace all of theCDRs with the complete CDRs from the donor variable region to transferthe antigen-binding capacity of one variable domain to another. Rather,it may only be necessary to transfer those residues that are necessaryto maintain the activity of the antigen-binding site. Given theexplanations set forth in U.S. Pat. Nos. 5,585,089, 5,693,761 and5,693,762, it will be well within the competence of those skilled in theart, either by carrying out routine experimentation or by trial anderror testing to obtain a functional antibody with reducedimmunogenicity.

Alterations to the variable region notwithstanding, those skilled in theart will appreciate that the modified antibodies of this invention willcomprise antibodies (e.g., full-length antibodies or immunoreactivefragments thereof) in which at least a fraction of one or more of theconstant region domains has been deleted or otherwise altered so as toprovide desired biochemical characteristics such as increased tumorlocalization or reduced serum half-life when compared with an antibodyof approximately the same immunogenicity comprising a native orunaltered constant region. In some embodiments, the constant region ofthe modified antibodies will comprise a human constant region.Modifications to the constant region compatible with this inventioncomprise additions, deletions or substitutions of one or more aminoacids in one or more domains. That is, the modified antibodies disclosedherein can comprise alterations or modifications to one or more of thethree heavy chain constant domains (CH1, CH2, or CH3) and/or to thelight chain constant domain (CL). In some embodiments, modified constantregions wherein one or more domains are partially or entirely deletedare contemplated. In some embodiments, the modified antibodies willcomprise domain deleted constructs or variants wherein the entire CH2domain has been removed (ACH2 constructs). In some embodiments, theomitted constant region domain will be replaced by a short amino acidspacer (e.g., 10 residues) that provides some of the molecularflexibility typically imparted by the absent constant region.

It will be noted that in certain embodiments, the modified antibodiescan be engineered to fuse the CH3 domain directly to the hinge region ofthe respective modified antibodies. In other constructs it may bedesirable to provide a peptide spacer between the hinge region and themodified CH2 and/or CH3 domains. For example, compatible constructscould be expressed wherein the CH2 domain has been deleted and theremaining CH3 domain (modified or unmodified) is joined to the hingeregion with a 5-20 amino acid spacer. Such a spacer can be added, forinstance, to ensure that the regulatory elements of the constant domainremain free and accessible or that the hinge region remains flexible.However, it should be noted that amino acid spacers can, in some cases,prove to be immunogenic and elicit an unwanted immune response againstthe construct. Accordingly, in certain embodiments, any spacer added tothe construct will be relatively non-immunogenic, or even omittedaltogether, so as to maintain the desired biochemical qualities of themodified antibodies.

Besides the deletion of whole constant region domains, it will beappreciated that the antibodies of the present invention can be providedby the partial deletion or substitution of a few or even a single aminoacid. For example, the mutation of a single amino acid in selected areasof the CH2 domain may be enough to substantially reduce Fc binding andthereby increase tumor localization. Similarly, it may be desirable tosimply delete that part of one or more constant region domains thatcontrol the effector function (e.g., complement C1Q binding) to bemodulated. Such partial deletions of the constant regions can improveselected characteristics of the antibody (serum half-life) while leavingother desirable functions associated with the subject constant regiondomain intact. Moreover, as alluded to above, the constant regions ofthe disclosed antibodies can be modified, e.g., through the mutation orsubstitution of one or more amino acids, which may enhance the profileof the resulting construct. In this respect it may be possible todisrupt the activity provided by a conserved binding site (e.g., Fcbinding) while substantially maintaining the configuration andimmunogenic profile of the modified antibody. Certain embodiments cancomprise the addition of one or more amino acids to the constant regionto enhance desirable characteristics such as decreasing or increasingeffector function or provide for more cytotoxin or carbohydrateattachment. In such embodiments it can be desirable to insert orreplicate specific sequences derived from selected constant regiondomains.

The present invention further embraces variants and equivalents whichare substantially homologous to the chimeric, humanized and humanantibodies, or antibody fragments thereof, set forth herein. These cancontain, for example, conservative substitution mutations, i.e., thesubstitution of one or more amino acids by similar amino acids. Forexample, conservative substitution refers to the substitution of anamino acid with another within the same general class such as, forexample, one acidic amino acid with another acidic amino acid, one basicamino acid with another basic amino acid or one neutral amino acid byanother neutral amino acid. What is intended by a conservative aminoacid substitution is well known in the art, such as those definedhereinabove.

The polypeptides of the present invention can be recombinantpolypeptides, natural polypeptides, or synthetic polypeptides comprisingan antibody, or fragment thereof, against a human CD123/IL-3Rα. It willbe recognized in the art that some amino acid sequences of the inventioncan be varied without significant effect of the structure or function ofthe protein. Thus, the invention further includes variations of thepolypeptides which show substantial activity or which include regions ofan antibody, or fragment thereof, against a CD123 antigen, such as ahuman CD123. Such mutants include deletions, insertions, inversions,repeats, and type substitutions.

The polypeptides and analogs can be further modified to containadditional chemical moieties not normally part of the protein. Thosederivatized moieties may improve the solubility, the biological halflife or absorption of the protein. The moieties can also reduce oreliminate any desirable side effects of the proteins and the like. Anoverview for those moieties can be found in REMINGTON'S PHARMACEUTICALSCIENCES, 20th ed., Mack Publishing Co., Easton, Pa. (2000).

The isolated polypeptides described herein can be produced by anysuitable method known in the art. Such methods range from direct proteinsynthetic methods to constructing a DNA sequence encoding isolatedpolypeptide sequences and expressing those sequences in a suitabletransformed host. In some embodiments, a DNA sequence is constructedusing recombinant technology by isolating or synthesizing a DNA sequenceencoding a wild-type protein of interest. Optionally, the sequence canbe mutagenized by site-specific mutagenesis to provide functionalanalogs thereof. See, e.g., Zoeller et al., Proc. Nat'l. Acad. Sci. USA81:5662-5066, 1984, and U.S. Pat. No. 4,588,585.

In some embodiments a DNA sequence encoding a polypeptide of interest(e.g., antibody, antigen-binding fragment, or polypeptide of theinvention) would be completely or partially constructed by chemicalsynthesis using an oligonucleotide synthesizer. Such oligonucleotidescan be designed based on the amino acid sequence of the desiredpolypeptide and selecting those codons that are favored in the host cellin which the recombinant polypeptide of interest will be produced.Standard methods can be applied to synthesize an isolated polynucleotidesequence encoding an isolated polypeptide of interest. For example, acomplete amino acid sequence can be used to construct a back-translatedgene. Further, a DNA oligomer containing a nucleotide sequence codingfor the particular isolated polypeptide can be synthesized. For example,several small oligonucleotides coding for portions of the desiredpolypeptide can be synthesized and then ligated. The individualoligonucleotides typically contain 5′ or 3′ overhangs for complementaryassembly.

Once assembled (by synthesis, site-directed mutagenesis or anothermethod), the polynucleotide sequences encoding a particular isolatedpolypeptide of interest will be inserted into an expression vector andoperatively linked to an expression control sequence appropriate forexpression of the protein in a desired host. Proper assembly can beconfirmed by nucleotide sequencing, restriction mapping, and expressionof a biologically active polypeptide in a suitable host. As is wellknown in the art, in order to obtain high expression levels of atransfected gene in a host, the gene must be operatively linked totranscriptional and translational expression control sequences that arefunctional in the chosen expression host.

In certain embodiments, recombinant expression vectors are used toamplify and express DNA encoding antibodies, or fragments thereof,against human CD123/IL-3Rα. Recombinant expression vectors arereplicable DNA constructs which have synthetic or cDNA-derived DNAfragments encoding a polypeptide chain of an anti-CD123/IL-3Rα antibody,or fragment thereof, operatively linked to suitable transcriptional ortranslational regulatory elements derived from mammalian, microbial,viral or insect genes. A transcriptional unit generally comprises anassembly of (1) a genetic element or elements having a regulatory rolein gene expression, for example, transcriptional promoters or enhancers,(2) a structural or coding sequence which is transcribed into mRNA andtranslated into protein, and (3) appropriate transcription andtranslation initiation and termination sequences. Such regulatoryelements can include an operator sequence to control transcription. Theability to replicate in a host, usually conferred by an origin ofreplication, and a selection gene to facilitate recognition oftransformants can additionally be incorporated. DNA regions areoperatively linked when they are functionally related to each other. Forexample, DNA for a signal peptide (secretory leader) is operativelylinked to DNA for a polypeptide if it is expressed as a precursor whichparticipates in the secretion of the polypeptide; a promoter isoperatively linked to a coding sequence if it controls the transcriptionof the sequence; or a ribosome binding site is operatively linked to acoding sequence if it is positioned so as to permit translation.Structural elements intended for use in yeast expression systems includea leader sequence enabling extracellular secretion of translated proteinby a host cell. Alternatively, where recombinant protein is expressedwithout a leader or transport sequence, it can include an N-terminalmethionine residue. This residue can optionally be subsequently cleavedfrom the expressed recombinant protein to provide a final product.

The choice of expression control sequence and expression vector willdepend upon the choice of host. A wide variety of expression host/vectorcombinations can be employed. Useful expression vectors for eukaryotichosts, include, for example, vectors comprising expression controlsequences from SV40, bovine papilloma virus, adenovirus andcytomegalovirus. Useful expression vectors for bacterial hosts includeknown bacterial plasmids, such as plasmids from Escherichia coli,including pCR 1, pBR322, pMB9 and their derivatives, wider host rangeplasmids, such as M13 and filamentous single-stranded DNA phages.

Suitable host cells for expression of a CD123/IL-3Rα-binding polypeptideor antibody (or a CD123/IL-3Rα protein to use as an antigen) includeprokaryotes, yeast, insect or higher eukaryotic cells under the controlof appropriate promoters. Prokaryotes include gram negative or grampositive organisms, for example E. coli or bacilli. Higher eukaryoticcells include established cell lines of mammalian origin, such as CHOcells. Cell-free translation systems could also be employed. Appropriatecloning and expression vectors for use with bacterial, fungal, yeast,and mammalian cellular hosts are described by Pouwels et al. (CloningVectors: A Laboratory Manual, Elsevier, N.Y., 1985), the relevantdisclosure of which is hereby incorporated by reference. Additionalinformation regarding methods of protein production, including antibodyproduction, can be found, e.g., in U.S. Patent Publication No.2008/0187954, U.S. Pat. Nos. 6,413,746 and 6,660,501, and InternationalPatent Publication No. WO 04009823, each of which is hereby incorporatedby reference herein in its entirety.

Various mammalian or insect cell culture systems are also advantageouslyemployed to express recombinant protein. Expression of recombinantproteins in mammalian cells can be performed because such proteins aregenerally correctly folded, appropriately modified and completelyfunctional. Examples of suitable mammalian host cell lines includeHEK-293 and HEK-293T, the COS-7 lines of monkey kidney cells, describedby Gluzman (Cell 23: 175, 1981), and other cell lines including, forexample, L cells, CI 27, 3T3, Chinese hamster ovary (CHO), HeLa and BHKcell lines. Mammalian expression vectors can comprise nontranscribedelements such as an origin of replication, a suitable promoter andenhancer linked to the gene to be expressed, and other 5′ or 3′ flankingnontranscribed sequences, and 5′ or 3′ nontranslated sequences, such asnecessary ribosome binding sites, a polyadenylation site, splice donorand acceptor sites, and transcriptional termination sequences.Baculovirus systems for production of heterologous proteins in insectcells are reviewed by Luckow and Summers, Bio/Technology 6:47 (1988).

Thus one aspect of the invention also provides a cell producing any oneof the subject antibody or antigen-binding fragment thereof, or any oneof the subject polypeptide. In certain embodiments, the cell is amammalian cell. In certain embodiments, the cell is a HEK-293 orHEK-293T cell, a COS-7 cell, an L cell, a CI 27 cell, a 3T3 cell, aChinese hamster ovary (CHO) cell, a HeLa cell, or a BHK cell. In certainembodiments, the cell is a CHO cell.

The proteins produced by a transformed host can be purified according toany suitable method. Such standard methods include chromatography (e.g.,ion exchange, affinity and sizing column chromatography),centrifugation, differential solubility, or by any other standardtechnique for protein purification. Affinity tags such as hexahistidine,maltose binding domain, influenza coat sequence andglutathione-S-transferase can be attached to the protein to allow easypurification by passage over an appropriate affinity column. Isolatedproteins can also be physically characterized using such techniques asproteolysis, nuclear magnetic resonance and x-ray crystallography.

For example, supernatants from systems which secrete recombinant proteininto culture media can be first concentrated using a commerciallyavailable protein concentration filter, for example, an Amicon orMillipore Pellicon ultrafiltration unit. Following the concentrationstep, the concentrate can be applied to a suitable purification matrix.Alternatively, an anion exchange resin can be employed, for example, amatrix or substrate having pendant diethylaminoethyl (DEAE) groups. Thematrices can be acrylamide, agarose, dextran, cellulose or other typescommonly employed in protein purification. Alternatively, a cationexchange step can be employed. Suitable cation exchangers includevarious insoluble matrices comprising sulfopropyl or carboxymethylgroups. Finally, one or more reversed-phase high performance liquidchromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media,e.g., silica gel having pendant methyl or other aliphatic groups, can beemployed to further purify a CD123/IL-3Rα-binding agent. Some or all ofthe foregoing purification steps, in various combinations, can also beemployed to provide a homogeneous recombinant protein.

Recombinant protein produced in bacterial culture can be isolated, forexample, by initial extraction from cell pellets, followed by one ormore concentration, salting-out, aqueous ion exchange or size exclusionchromatography steps. High performance liquid chromatography (HPLC) canbe employed for final purification steps. Microbial cells employed inexpression of a recombinant protein can be disrupted by any convenientmethod, including freeze-thaw cycling, sonication, mechanicaldisruption, or use of cell lysing agents.

Methods known in the art for purifying antibodies and other proteinsalso include, for example, those described in U.S. Patent PublicationNos. 2008/0312425, 2008/0177048, and 2009/0187005, each of which ishereby incorporated by reference herein in its entirety.

In certain embodiments, the CD123/IL-3Rα-binding agent of the presentinvention have a N-terminal serine, which can be oxidized with anoxidizing agent to form an oxidized CD123/IL-3Rα-binding agent having aN-terminal aldehyde group.

Any suitable oxidizing agent can be used in step (a) of the methodsdescribed above. In certain embodiments, the oxidizing agent is aperiodate. More specifically, the oxidizing agent is sodium periodate.

Excess molar equivalents of the oxidizing agent relative to theCD123/IL-3Rα-binding agent can be used. In certain embodiments, about2-100, 5-80, 10-50, 1-10 or 5-10 molar equivalents of the oxidizingagent can be used. In certain embodiments, about 10 or about 50equivalents of the oxidizing agent can be used. When large amount of theoxidizing agent is used, short reaction time is used to avoidover-oxidation. For example, when 50 equivalents of the oxidizing agentis used, the oxidation reaction is carried out for about 5 to about 60minutes. Alternatively, when 10 equivalents of the oxidizing agent isused, the reaction is carried out for about 30 minutes to about 24hours. In one embodiment, 5-10 molar equivalents of the oxidizing agentis used and the oxidation reaction is carried out for about 5 to about60 minutes (e.g., about 10 to about 30 minutes, about 20 to about 30minutes).

In certain embodiments, the oxidation reaction does not lead tosignificant non-targeted oxidation. For example, no signification extent(e.g., less than 20%, less than 10%, less than 5%, less than 3%, lessthan 2% or less than 1%) of methionine and/or glycans are oxidizedduring the oxidation process of N-terminal serine to generate theoxidized CD123/IL-3Rα-binding agent having a N-terminal aldehyde group.

In certain embodiments, the CD123/IL-3Rα-binding agent of the presentinvention have a recombinantly engineered Cys residue, such as a Cysresidue corresponding to the 5^(th) to the last Cys in, for example, SEQID NO: 54 or 56 (i.e, a Cys residue at EU/OU numbering position 442).Thus the term “cysteine engineered antibody” includes an antibody withat least one Cys that is not normally present at a given residue of theantibody light chain or heavy chain. Such Cys, which may also bereferred to as “engineered Cys,” can be engineered using anyconventional molecular biology or recombinant DNA technology (e.g., byreplacing the coding sequence for a non-Cys residue at the targetresidue with a coding sequence for Cys). For example, if the originalresidue is Ser with a coding sequence of 5′-UCU-3′, the coding sequencecan be mutated (e.g., by site-directed mutagenesis) to 5′-UGU-3′, whichencodes Cys. In certain embodiments, the Cys engineered antibody of theinvention has an engineered Cys in the heavy chain. In certainembodiments, the engineered Cys is in or near the CH3 domain of theheavy chain. In certain embodiments, the engineered Cys corresponding tothe 5^(th) to the last Cys in, for example, SEQ ID NO: 54 or 56. Theengineered antibody heavy (or light) chain sequence can be inserted intoa suitable recombinant expression vector to produce the engineeredantibody having the engineered Cys residue in place of the original Serresidue.

3. Immunoconjugates

In a second aspect, the present invention also provides immunoconjugatescomprising CD123/IL-3Rα-binding agents described herein covalentlylinked to one or more molecules of the cytototoxic agents describedherein.

In a first embodiments, the immunoconjugate of the present inventioncomprises a CD123/IL-3Rα-binding agents (including antibody,antigen-binding fragment thereof, or polypeptide comprising the antibodyor antigen-binding fragment thereof) described herein covalently linkedto a cytotoxic agent described herein through the ε-amino group of oneor more lysine residues located on the CD123/IL-3Rα-binding agents.

In a 1^(st) specific embodiment of the first embodiment, theimmunoconjugate of the present invention is represented by the followingformula:CBA

Cy^(L1))_(W) _(L)   (L1),wherein:

CBA is a CD123/IL-3Rα-binding agent (e.g. a subject antibody orantigen-binding fragment thereof described herein above, or a subjectpolypeptide thereof described above), that is covalently linked througha lysine residue to Cy^(L1);

W_(L) is an integer from 1 to 20; and

Cy^(L1) is a cytotoxic compound represented by the following formula:

or a pharmaceutically acceptable salt thereof, wherein:

the double line

between N and C represents a single bond or a double bond, provided thatwhen it is a double bond, X is absent and Y is —H or a (C₁-C₄)alkyl; andwhen it is a single bond, X is —H or an amine protecting moiety, and Yis —OH or —SO₃M;

W′ is —NR^(e′),

R^(e′) is —(CH₂—CH₂—O)_(n)R^(k);

n is an integer from 2 to 6;

R^(k) is —H or -Me;

R^(x3) is a (C₁-C₆)alkyl;

L′ is represented by the following formula:—NR₅—P—C(═O)—(CR_(a)R_(b))_(m)—C(═O)—  (B1′); or—NR₅—P—C(═O)—(CR_(a)R_(b))_(m)—S—Z^(s1)—  (B2′);

R₅ is —H or a (C₁-C₃)alkyl;

P is an amino acid residue or a peptide containing between 2 to 20 aminoacid residues;

R_(a) and R_(b), for each occurrence, are each independently —H,(C₁-C₃)alkyl, or a charged substituent or an ionizable group Q;

m is an integer from 1 to 6; and

Z^(s1) is selected from any one of the following formulas:

wherein:

q is an integer from 1 to 5; and

M is H⁺ or a cation.

In a 2^(nd) specific embodiment, for conjugates of formula (L1), Cy^(L1)is represented by formula (L1a) or (L1a1); and the remaining variablesare as described above in the 1^(st) specific embodiment.

In a 3^(rd) specific embodiment, for conjugates of formula (L1), Cy^(L1)is represented by formula (L1b) or (L1b1); and the remaining variablesare as described above in the 1^(st) specific embodiment. Morespecifically, R^(x3) is a (C₂-C₄)alkyl.

In a 4^(th) specific embodiment, for conjugates of formula (L1), Cy^(L1)is represented by formula (L1a); R_(a) and R_(b) are both H; R₅ is H orMe, and the remaining variables are as described above in the 1^(st)specific embodiment.

In a 5^(th) specific embodiment, P is a peptide containing 2 to 5 aminoacid residues; and the remaining variables are described above in the1^(st), 2^(nd) or 4^(th) specific embodiment. In a more specificembodiment, P is selected from the group consisting of Gly-Gly-Gly,Ala-Val, Val-Ala, Val-Cit, Val-Lys, Phe-Lys, Lys-Lys, Ala-Lys, Phe-Cit,Leu-Cit, Ile-Cit, Trp, Cit, Phe-Ala, Phe-N⁹-tosyl-Arg, Phe-N⁹-nitro-Arg,Phe-Phe-Lys, D-Phe-Phe-Lys, Gly-Phe-Lys, Leu-Ala-Leu, Ile-Ala-Leu,Val-Ala-Val, Ala-Leu-Ala-Leu (SEQ ID NO: 55), β-Ala-Leu-Ala-Leu (SEQ IDNO: 57), Gly-Phe-Leu-Gly (SEQ ID NO: 73), Val-Arg, Arg-Val, Arg-Arg,Val-D-Cit, Val-D-Lys, Val-D-Arg, D-Val-Cit, D-Val-Lys, D-Val-Arg,D-Val-D-Cit, D-Val-D-Lys, D-Val-D-Arg, D-Arg-D-Arg, Ala-Ala, Ala-D-Ala,D-Ala-Ala, D-Ala-D-Ala, Ala-Met, and Met-Ala. More specifically, P isGly-Gly-Gly, Ala-Val, Ala-Ala, Ala-D-Ala, D-Ala-Ala, or D-Ala-D-Ala.

In a 6^(th) specific embodiment, Q is —SO₃M; and the remaining variablesare as described above in the 1^(st), 2^(nd), 4^(th) or 5^(th) specificembodiment or any more specific embodiments described therein.

In a 7^(th) specific embodiment, the immunoconjugate of the firstembodiment is represented by the following formula:

or a pharmaceutically acceptable salt thereof, wherein W_(L) is aninteger from 1 to 10; the double line

between N and C represents a single bond or a double bond, provided thatwhen it is a double bond, X is absent and Y is —H; and when it is asingle bond, X is —H, and Y is —OH or —SO₃M. In a more specificembodiment, the double line

between N and C represents a double bond, X is absent and Y is —H. Inanother more specific embodiment, the double line

between N and C represents a single bond, X is —H and Y is —SO₃M.

In a 8^(th) specific embodiment, the immunoconjugate of the firstembodiment is represented by the following formula:CBA

Cy^(L2))_(W) _(L)   (L2),wherein:

CBA is a CD123/IL-3Rα-binding agent described in the first aspect of thepresent invention (e.g. a subject antibody or antigen-binding fragmentthereof described herein above, or a subject polypeptide thereofdescribed above), that is covalently linked to Cy^(L2) through a lysineresidue;

W_(L) is an integer from 1 to 20; and

Cy^(L2) is a cytotoxic compound represented by the following formula:

or a pharmaceutically acceptable salt thereof, wherein:

the double line

between N and C represents a single bond or a double bond, provided thatwhen it is a double bond, X is absent and Y is —H or a (C₁-C₄)alkyl; andwhen it is a single bond, X is —H or an amine protecting moiety, and Yis —OH or —SO₃M;

R^(x1) and R^(x2) are independently (C₁-C₆)alkyl;

R^(e) is —H or a (C₁-C₆)alkyl;

W′ is —NR^(e′),

R^(e′) is —(CH₂—CH₂—O)_(n)—R^(k);

n is an integer from 2 to 6;

R^(k) is —H or -Me;

Z^(s1) is selected from any one of the following formulas:

wherein:

q is an integer from 1 to 5; and

M is —H⁺ or a cation.

In a 9^(th) specific embodiment, for immunoconjugates of formula (L2),Cy^(L2) is represented by formula (L2a) or (L2a1); and the remainingvariables are as described above in the 8^(th) specific embodiment.

In a 10^(th) specific embodiment, for immunoconjugates of formula (L2),Cy^(L2) is represented by formula (L2b) or (L2b1); and the remainingvariables are as described above in the 8^(th) specific embodiment.

In a 11^(th) specific embodiment, for immunoconjugates of formula (L2),R^(e) is H or Me; R^(x1) and R^(x2) are independently—(CH₂)_(p)—(CR^(f)R^(g))—, wherein R^(f) and R^(g) are eachindependently —H or a (C₁-C₄)alkyl; and p is 0, 1, 2 or 3; and theremaining variables are as described above in the 8^(th), 9^(th) or10^(th) specific embodiment. More specifically, R^(f) and R^(g) are thesame or different, and are selected from —H and -Me.

In a 12^(th) specific embodiment, the immunoconjugate of the firstembodiment is represented by the following formula:

or a pharmaceutically acceptable salt thereof, wherein W_(L) is aninteger from 1 to 10; the double line

between N and C represents a single bond or a double bond, provided thatwhen it is a double bond, X is absent and Y is —H; and when it is asingle bond, X is —H and Y is —OH or —SO₃M. In a more specificembodiment, the double line

between N and C represents a double bond. In another more specificembodiment, the double line

between N and C represents a single bond, X is —H and Y is —SO₃M.

In a 13^(th) specific embodiment, the immunoconjugates of the firstembodiment is represented by the following formula:CBA

Cy^(L3))_(W) _(L)   (L3),wherein:

CBA is a CD123/IL-3Rα-binding agents described in the first aspect ofthe present invention (e.g. a subject antibody or antigen-bindingfragment thereof described herein above, or a subject polypeptidethereof described above), which is covalently linked to Cy^(L3) througha Lys residue;

W_(L) is an integer from 1 to 20;

Cy^(L3) is represented by the following formula:

m′ is 1 or 2;

R₁ and R₂, are each independently H or a (C₁-C₃)alkyl; and

Z^(s1) is selected from any one of the following formulas:

wherein:

q is an integer from 1 to 5; and

M is H⁺ or a cation.

In a 14^(th) specific embodiment, for immunoconjugates of formula (L3),m′ is 1, and R₁ and R₂ are both H; and the remaining variables are asdescribed above in the 13^(th) specific embodiment.

In a 15^(th) specific embodiment, for immunoconjugates of formula (L3),m′ is 2, and R₁ and R₂ are both Me; and the remaining variables are asdescribed above in the 13^(th) specific embodiment.

In a 16^(th) specific embodiment, the immunoconjugates of the firstembodiment is represented by the following formula:

or a pharmaceutically acceptable salt thereof, wherein W_(L) is aninteger from 1 to 10.

In a 17^(th) specific embodiment, for immunoconjugates of the firstembodiment, M is H⁺, Na⁺ or K⁺; and the remaining variables are asdescribed above in any one of the 1^(st) to 16^(th) specific embodimentor any more specific embodiments described therein.

In any of the above 1^(st) to the 17^(th) specific embodiments, thesubject antibody or antigen-binding fragment thereof may have one ormore of (e.g., substantially all of, or 100% of) the Lys residues in anyof the six light chain and heavy chain CDR regions (if any) substitutedby Arg. The subject antibody or antigen-binding fragment thereof maycomprise an immunoglobulin heavy chain variable region (HCVR) having theamino acid sequence set forth in SEQ ID NO: 39 or 40; and animmunoglobulin light chain variable region (LCVR) having the amino acidsequence set forth in SEQ ID NO: 41. The subject antibody orantigen-binding fragment thereof may also comprise an Ig HCVR having theamino acid sequence set forth in SEQ ID NO: 34; and an Ig LCVR havingthe amino acid sequence set forth in SEQ ID NO: 35. The subject antibodyor antigen-binding fragment thereof may further comprise an Ig HCVRhaving the amino acid sequence set forth in SEQ ID NO: 32, 34, 38, 39,or 40; and an Ig LCVR having the amino acid sequence set forth in SEQ IDNO: 33, 35, 37, or 41. In certain embodiments, the second residue fromthe N-terminus of SEQ ID NO: 34 is Phe, while in certain otherembodiments, the second residue from the N-terminus of SEQ ID NO: 34 isVal.

The immunoconjugates described the first embodiment or any specificembodiments descried therein can be prepared according to any methodsknown in the art, see, for example, WO 2012/128868 and WO2012/112687,which are incorporate herein by reference.

In certain embodiments, the immunoconjugates of the first embodiment canbe prepared by a first method comprising the steps of reacting the CBAwith a cytotoxic agent having an amine reactive group.

In one embodiment, for the first method described above, the reaction iscarried out in the presence of an imine reactive reagent, such asNaHSO₃.

In one embodiment, for the first method described above the cytotoxicagent having an amine reactive reagent is represented by the followingformula:

or a pharmaceutically acceptable salt thereof, wherein:

L^(c) is represented by the following formula:—NR₅—P—C(═O)—(CR_(a)R_(b))_(m)—C(═O)E  (B1); or—NR₅—P—C(═O)—(CR_(a)R_(b))_(m)—S—Z^(s)  (B2)

C(═O)E is a reactive ester group, such as N-hydroxysuccinimide ester,N-hydroxy sulfosuccinimide ester, nitrophenyl (e.g., 2 or 4-nitrophenyl)ester, dinitrophenyl (e.g., 2,4-dinitrophenyl) ester,sulfo-tetrafluorophenyl (e.g., 4-sulfo-2,3,5,6-tetrafluorophenyl) ester,or pentafluorophenyl ester, preferably N-hydroxysuccinimide ester;

Z^(s) is represented by the following formula:

wherein:

-   -   q is an integer from 1 to 5; and    -   U is —H or SO₃M; and        the remaining variables are as described in any one of the        1^(st) to 7^(th) and 17^(th) specific embodiments or any more        specific embodiments described therein.

In certain embodiments, the immunoconjugates of the first embodiment canbe prepared by a second method comprising the steps of:

(a) reacting a cytotoxic agent with a linker compound having an aminereactive group and a thiol reactive group to form a cytotoxicagent-linker compound having the amine reactive group bound thereto; and

(b) reacting the CBA with the cytotoxic agent-linker compound.

In one embodiment, for the second method described above, the reactionin step (a) is carried out in the presence of an imine reactive reagent.

In one embodiment, for the second method described above, the cytotoxicagent-linker compound is reacted with the CBA without purification.Alternatively, the cytotoxic agent-linker compound is first purifiedbefore reacting with the CBA.

In certain embodiments, the immunoconjugates of the first embodiment canbe prepared by a third method comprising the steps of:

(a) reacting the CBA with a linker compound having an amine reactivegroup and a thiol reactive group to form a modified CBA having a thiolreactive group bound thereto; and

(b) reacting the modified CBA with the cytotoxic agent.

In one embodiment, for the third method described above, the reaction instep (b) is carried out in the presence of an imine reactive reagent.

In certain embodiments, the immunoconjugates of the first embodiment canbe prepared by a fourth method comprising the steps of reacting the CBA,a cytotoxic compound and a linker compound having an amine reactivegroup and a thiol reactive group.

In one embodiment, for the fourth method, the reaction is carried out inthe presence of an imine reactive agent.

In certain embodiments, for the second, third or fourth embodiment,described above, the linker compound having an amine reactive group anda thiol reactive group is represented by the following formula:

wherein X is halogen; J_(D)-SH, —SSR^(d), or —SC(═O)R^(g); R^(d) isphenyl, nitrophenyl, dinitrophenyl, carboxynitrophenyl, pyridyl ornitropyridyl; R^(g) is an alkyl; and the remaining variables are asdescribed above for formula (a1)-(a10); and the cytotoxic agent isrepresented by the following formula:

or a pharmaceutically acceptable salt thereof, wherein the variables areas described in any one of the 8^(th) to 12^(th) and 17^(th) specificembodiments and any more specific embodiments described therein.

In certain embodiments, for the second, third or fourth methodsdescribed above, the linker compound having an amine reactive group anda thiol reactive group is represented by any one of the formula(a1L)-(a10L) and the cytotoxic agent is represented by the followingformula:

Wherein the variables are as described above in any one of the 13^(th)to 17^(th) specific embodiments and any more specific embodimentsdescribed therein.

In a second embodiment, the immuonoconjugate of the present inventioncomprises an oxidized CD123/IL-3Rα-binding agent (including antibody,antigen-binding fragment thereof, or polypeptide comprising the antibodyor antigen-binding fragment thereof) described in the first aspect ofthe present invention described herein (e.g., oxidized antibody orantigen-binding fragment thereof, or the polypeptide thereof) covalentlylinked to a cytotoxic agent described herein through one or morealdehyde groups located on the oxidized CD123-binding agent. Thealdehyde groups located on the oxidized CD123/IL-3Rα-binding agent canbe generated by oxidizing one or more 2-hydroxyethylamine moiety of theCD123/IL-3Rα-binding agent, wherein the 2-hydroxyethylamine moiety ispart of a serine, threonine, hydroxylysine, 4-hydroxyornithie or2,4-diamino-5-hydroxy valeric acid residue. In one embodiment, thealdehyde groups can be generated by oxidizing the 2-hydroxyethylaminemoiety of one or more N-terminal serine residue(s) located on theCD123/IL-3Rα-binding agent (e.g., 1, 2, 3, or up to 4 Ser residues atthe N-termini of the light chains and/or the heavy chains).

In a 1^(st) specific embodiment of the second embodiment, theimmunoconjugate of the present invention is represented by the followingformula:

wherein:

CBA is the oxidized CD123/IL-3Rα-binding agent described in the firstaspect of the invention (e.g. a subject oxidized antibody orantigen-binding fragment thereof described herein above, or a subjectoxidized polypeptide thereof described above);

W_(S) is 1, 2, 3, or 4;

J_(CB)′ is a moiety formed by reacting an aldehyde group on the CBA withan aldehyde reactive group on Cy^(s1), and is represented by thefollowing formula:

wherein s1 is the site covalently linked to the CBA; and s2 is the sitecovalently linked to Cy^(s1);

Cy^(s1) is represented by the following formula:

or a pharmaceutically acceptable salt thereof, wherein:

the double line

between N and C represents a single bond or a double bond, provided thatwhen it is a double bond, X is absent and Y is —H or a (C₁-C₄)alkyl; andwhen it is a single bond, X is —H or an amine protecting moiety, Y is—OH or —SO₃M, and M is H⁺ or a cation;

R₅ is —H or a (C₁-C₃)alkyl;

P is an amino acid residue or a peptide containing 2 to 20 amino acidresidues;

Z_(d1) is absent, —C(═O)—NR₉—, or —NR₉—C(═O)—;

R₉ is —H or a (C₁-C₃)alkyl;

R_(a) and R_(b), for each occurrence, are independently —H,(C₁-C₃)alkyl, or a charged substituent or an ionizable group Q;

r and r′ are independently an integer from 1 to 6;

W′ is —NR^(e′),

R^(e′) is —(CH₂—CH₂—O)_(n)—R^(k);

n is an integer from 2 to 6;

R^(k) is —H or -Me;

R^(x3) is a (C₁-C₆)alkyl;

L is —NR₉—(CR_(a)R_(b))_(r″) or absent; and

r″ is an integer from 0 to 6.

In a 2^(nd) specific embodiment, for immunoconjugates of formula (S1),Cy^(s1) is represented by formula (S1a) or (S1a1); and the remainingvariables are as described above in the 1^(st) specific embodiment.

In a 3^(rd) specific embodiment, for immunoconjugates of formula (S1),Cy^(s1) is represented by formula (S1b) or (S1b1); and the remainingvariables are as described above in the 1^(st) specific embodiment. Morespecifically, R^(x3) is a (C₂-C₄)alkyl.

In a 4^(th) specific embodiment, for immunoconjugates of formula (S1),R_(a) and R_(b) are both H, and R₅ and R₉ are both H or Me; and theremaining variables are as described above in the 1^(st) or 2^(nd)specific embodiment.

In a 5^(th) specific embodiment, for immunoconjugates of formula (S1), Pis a peptide containing 2 to 5 amino acid residues; and the remainingvariables are as described above in the 1^(st), 2^(nd) or 4^(th)specific embodiment. In a more specific embodiment, P is selected fromthe group consisting of Gly-Gly-Gly, Ala-Val, Val-Ala, Val-Cit, Val-Lys,Phe-Lys, Lys-Lys, Ala-Lys, Phe-Cit, Leu-Cit, Ile-Cit, Trp, Cit, Phe-Ala,Phe-N⁹-tosyl-Arg, Phe-N⁹-nitro-Arg, Phe-Phe-Lys, D-Phe-Phe-Lys,Gly-Phe-Lys, Leu-Ala-Leu, Ile-Ala-Leu, Val-Ala-Val, Ala-Leu-Ala-Leu (SEQID NO: 55), β-Ala-Leu-Ala-Leu (SEQ ID NO: 57), Gly-Phe-Leu-Gly (SEQ IDNO: 73), Val-Arg, Arg-Val, Arg-Arg, Val-D-Cit, Val-D-Lys, Val-D-Arg,D-Val-Cit, D-Val-Lys, D-Val-Arg, D-Val-D-Cit, D-Val-D-Lys, D-Val-D-Arg,D-Arg-D-Arg, Ala-Ala, Ala-D-Ala, D-Ala-Ala, D-Ala-D-Ala, Ala-Met, andMet-Ala. Even more specifically, P is Gly-Gly-Gly, Ala-Val, Ala-Ala,Ala-D-Ala, D-Ala-Ala, or D-Ala-D-Ala.

In a 6^(th) specific embodiment, for immunoconjugates of formula (S1), Qis —SO₃M; and the remaining variables are as described above in the1^(st), 2^(nd), 4^(th) or 5^(th) specific embodiment.

In a 7^(th) specific embodiment, the immunoconjugate of the secondembodiment is represented by the following formula:

or a pharmaceutically acceptable salt thereof, wherein the double line

between N and C represents a single bond or a double bond, provided thatwhen it is a double bond, X is absent and Y is —H, and when it is asingle bond, X is —H, and Y is —OH or —SO₃M. In a more specificembodiment, the double line

between N and C represents a double bond, X is absent and Y is —H. Inanother more specific embodiment, the double line

between N and C represents a single bond, X is —H and Y is —SO₃M.

In an 8^(th) specific embodiment, the immunoconjugates of the presentinvention is represented by the following formula:

wherein:

CBA is the oxidized CD123/IL-3Rα-binding agent described in the firstaspect of the invention (e.g. a subject oxidized antibody orantigen-binding fragment thereof described herein above, or a subjectoxidized polypeptide thereof described above);

J_(CB)′ is a moiety formed by reacting an aldehyde group on the CBA andan aldehyde reactive group on Cy^(s2), and is represented by thefollowing formula:

wherein s1 is the site covalently linked to the CBA; and s2 is the sitecovalently linked to Cy^(s2);

Cy^(s2) is represented by the following formula:

or a pharmaceutically acceptable salt thereof, wherein:

the double line

between N and C represents a single bond or a double bond, provided thatwhen it is a double bond, X is absent and Y is —H or a (C₁-C₄)alkyl; andwhen it is a single bond, X is —H or an amine protecting moiety, and Yis —OH or —SO₃M;

M is H⁺ or a cation;

R^(x1) is a (C₁-C₆)alkyl;

R^(e) is —H or a (C₁-C₆)alkyl;

W′ is —NR^(e′),

R^(e′) is —(CH₂—CH₂—O)_(n)—R^(k);

n is an integer from 2 to 6;

R^(k) is —H or -Me;

R^(x2) is a (C₁-C₆)alkyl;

L₁ is represented by the following formula:

wherein:

s3 is the site covalently linked to the group J_(CB)′;

s4 is the site covalently linked to the —S— group on Cy^(s2);

Z_(a2) is absent, —C(═O)—NR₉—, or —NR₉—C(═O)—;

R₉ is —H or a (C₁-C₃)alkyl;

Q is H, a charged substituent or an ionizable group;

R_(a1), R_(a2), R_(a3), R_(a4), for each occurrence, are independently Hor (C₁-C₃)alkyl; and

q1 and r1 are each independently an integer from 0 to 10, provided thatq1 and r1 are not both 0.

In a more specific embodiment, Z_(a2) is absent; q1 and r1 are eachindependent an integer from 0 to 3, provided that q1 and r1 are not both0; and the remaining variables are as described above in the 8^(th)specific embodiments. Even more specifically, R_(a1), R_(a2), R_(a3),R_(a4) are all —H.

In another more specific embodiment, Z_(a2) is —C(═O)—NH—, or—NH₉—C(═O)—; q1 and r1 are each independently an integer from 1 to 6;and the remaining variables are as described above in the 8^(th)specific embodiments. Even more specifically, R_(a1), R_(a2), R_(a3),R_(a4) are all —H.

In a 9^(th) specific embodiment, for immunoconjugate of formula (S2),Cy^(s2) is represented by formula (S2a) or (S2a1); and the remainingvariables are as described above in the 8^(th) specific embodiment orany more specific embodiments described therein.

In a 10^(th) specific embodiment, for immunoconjugate of formula (S2),Cy^(s2) is represented by formula (S2b) or (S2b1); and the remainingvariables are as described above in the 8^(th) specific embodiment orany more specific embodiments described therein.

In an 11^(th) specific embodiment, for immunoconjugate of formula (S2),-L₁- is represented by the following formula:

-   -   or a pharmaceutically acceptable salt thereof, wherein R is H or        —SO₃M; and the remaining variables are as described above in the        8^(th), 9^(th) or 10^(th) specific embodiment or any more        specific embodiments described therein.

In a 12^(th) specific embodiment, for immunoconjugate of formula (S2),R^(e) is H or Me; and R^(x1) is —(CH₂)_(p)—(CR^(f)R^(g))—, and R^(x2) is—(CH₂)_(p)—(CR^(f)R^(g))—, wherein R^(f) and R^(g) are eachindependently —H or a (C₁-C₄)alkyl; and p is 0, 1, 2 or 3. Morespecifically, R^(f) and R^(g) are the same or different, and areselected from —H and -Me.

In a 13^(th) specific embodiment, the immunoconjugate of the secondembodiment is represented by the following formula:

-   -   or a pharmaceutically acceptable salt thereof, wherein the        double line        between N and C represents a single bond or a double bond,        provided that when it is a double bond, X is absent and Y is —H;        and when it is a single bond, X is —H; and Y is —OH or —SO₃M. In        a more specific embodiment, the double line        between N and C represents a double bond, X is absent and Y is        —H. In another more specific embodiment, the double line        between N and C represents a single bond, X is —H and Y is        —SO₃M.

In a 14^(th) specific embodiment, the immunoconjugate of the secondembodiment is represented by the following formula:

wherein:

CBA is the oxidized CD123/IL-3Rα-binding agent described in the firstaspect of the invention (e.g. a subject oxidized antibody orantigen-binding fragment thereof described herein above, or a subjectoxidized polypeptide thereof described above);

J_(CB)′ is a moiety formed by reacting an aldehyde group on the CBA andan aldehyde reactive group on Cy^(s3), and is represented by thefollowing formula:

wherein s1 is the site covalently linked to the CBA; and s2 is the sitecovalently linked to Cy^(s3);

Cy^(s3) is represented by the following formula:

wherein:

m′ is 1 or 2;

R₁ and R₂, are each independently H or a (C₁-C₃)alkyl;

L₁ is represented by the following formula:

wherein:

s3 is the site covalently linked to the group J_(CB)′;

s4 is the site covalently linked to the —S— group on Cy^(s3);

Z_(a2) is absent, —C(═O)—NR₉—, or —NR₉—C(═O)—;

R₉ is —H or a (C₁-C₃)alkyl;

Q is H, a charged substituent or an ionizable group;

R_(a1), R_(a2), R_(a3), R_(a4), for each occurrence, are independently Hor a (C₁-C₃)alkyl; and

q1 and r1 are each independently an integer from 0 to 10, provided thatq1 and r1 are not both 0.

In a more specific embodiment, Z_(a2) is absent; q1 and r1 are eachindependent an integer from 0 to 3, provided that q1 and r1 are not both0; and the remaining variables are as described above in the 14^(th)specific embodiments. Even more specifically, R_(a1), R_(a2), R_(a3),R_(a4) are all —H.

In another more specific embodiment, Z_(a2) is —C(═O)—NH—, or—NH₉—C(═O)—; q1 and r1 are each independently an integer from 1 to 6;and the remaining variables are as described above in the 14^(th)specific embodiments. Even more specifically, R_(a1), R_(a2), R_(a3),R_(a4) are all —H.

In a 15^(th) specific embodiment, for immunoconjugates of formula (S3),m′ is 1; R₁ and R₂ are both H; and the remaining variables are asdescribed above in the 14^(th) specific embodiment or any more specificembodiments described therein.

In a 16^(th) specific embodiment, for immunoconjugates of formula (S3),m′ is 2; R₁ and R₂ are both Me; and the remaining variables are asdescribed above in the 14^(th) specific embodiment or any more specificembodiments described therein.

In a 17^(th) specific embodiment, for immunoconjugates of formula (S3),-L₁- is represented by the following formula:

or a pharmaceutically acceptable salt thereof, wherein R is H or —SO₃Mand M is H⁺ or a cation.

In a 18^(th) specific embodiment, the immunoconjugate of the secondembodiment is represented by the following formula:

-   -   or a pharmaceutically acceptable salt thereof; wherein DM is        represented by the following formula:

In a 19^(th) specific embodiment, the immunoconjugate of the secondembodiment is represented by the following formula:

wherein:

CBA is the oxidized CD123/IL-3Rα-binding agent described in the firstaspect of the invention (e.g. a subject oxidized antibody orantigen-binding fragment thereof described herein above, or a subjectoxidized polypeptide thereof described above);

J_(CB)′ is a moiety formed by reacting an aldehyde group on the CBA andan aldehyde reactive group on Cy^(s4) and is represented by thefollowing formula:

wherein s1 is the site covalently linked to the CBA; and s2 is the sitecovalently linked to Cy^(s4);

Cy^(s4) is represented by the following formula:

L₁′ is represented by the following formula:

wherein:

s3 is the site covalently linked to the group J_(CB)′ group;

s4 is the site covalently linked to —NMe- group on Cy^(s4);

Z_(b1) and Z_(b2) are both absent, or one of Z_(b1) and Z_(b2) is absentand the other is —CH₂—O— or —O—CH₂—;

Z_(b1)′ and Z_(b2)′ are each independently absent, —CH₂—O—, —O—CH₂—,—NR₉—C(═O)—CH₂—, or —CH₂—C(═O)—NR₉—;

R₉ is H or (C₁-C₃)alkyl;

n1 and m1 are each independently an integer from 1 to 6;

one of E₁ and E₂ is —C(═O)—, and the other is —NR₉—; or one of E₁ and E₂is —C(═O)— or —NR₉—, and the other is absent;

P is an amino acid residue or a peptide containing between 2 to 20 aminoacid residues; and

R_(b1), R_(b2), R_(b3), R_(b4), R_(b5) and R_(b6), for each occurrence,are each independently H or a (C₁-C₃)alkyl.

In a 20^(th) specific embodiment, for immunoconjugates of formula (S4),R_(b1), R_(b2), R_(b3), R_(b4), R_(b5), and R_(b6) are all H; and theremaining variables are as described above in the 19^(th) specificembodiment.

In a 21^(st) specific embodiment, for immunoconjugates of formula (S4),R₉ is H; and the remaining variables are as described above in the19^(th) or 20^(th) specific embodiment.

In a 22^(nd) specific embodiment, for immunoconjugates of formula (S4),Z_(b1)′ and Z_(b2)′ are both absent; or Z_(b1)′ is —CH₂—O— and Z_(b2)′is absent; or Z_(b1)′ is —CH₂—C(═O)—NR₉—; and Z_(b2)′ is —O—CH₂— orabsent; and the remaining variables are as described above in the19^(th), 20^(th) or 21^(st) specific embodiment.

In a 23^(rd) specific embodiment, for immunoconjugates of formula (S4),P is a peptide containing 2 to 5 amino acid residues; and the remainingvariables are as described above in the 19^(th), 20^(th), 21^(st) or22^(nd) specific embodiment. In a more specific embodiment, P isselected from the group consisting of Gly-Gly-Gly, Ala-Val, Val-Ala,Val-Cit, Val-Lys, Phe-Lys, Lys-Lys, Ala-Lys, Phe-Cit, Leu-Cit, Ile-Cit,Trp, Cit, Phe-Ala, Phe-N⁹-tosyl-Arg, Phe-N⁹-nitro-Arg, Phe-Phe-Lys,D-Phe-Phe-Lys, Gly-Phe-Lys, Leu-Ala-Leu, Ile-Ala-Leu, Val-Ala-Val,Ala-Leu-Ala-Leu (SEQ ID NO: 55), β-Ala-Leu-Ala-Leu (SEQ ID NO: 57),Gly-Phe-Leu-Gly (SEQ ID NO: 73), Val-Arg, Arg-Val, Arg-Arg, Val-D-Cit,Val-D-Lys, Val-D-Arg, D-Val-Cit, D-Val-Lys, D-Val-Arg, D-Val-D-Cit,D-Val-D-Lys, D-Val-D-Arg, D-Arg-D-Arg, Ala-Ala, Ala-D-Ala, D-Ala-Ala,D-Ala-D-Ala, Ala-Met, and Met-Ala; and the remaining variables are asdescribed above in the 23^(rd) specific embodiment. Even morespecifically, P is Gly-Gly-Gly, Ala-Val, Ala-Ala, Ala-D-Ala, D-Ala-Ala,or D-Ala-D-Ala.

In a 24^(th) specific embodiment, the immunoconjugate of the secondembodiment is represented by the following formula:

-   -   or a pharmaceutically acceptable salt thereof, wherein DM is        represented by the following structural formula:

In a 25^(th) specific embodiment, for immunoconjugates of the secondembodiment, M is H⁺, Na⁺ or K⁺; and the remaining variables are asdescribed above in any one of the 1^(st) to 24^(th) specific embodimentor any more specific embodiments described therein.

In any of the above 1^(st) to the 25^(th) specific embodiments, thesubject oxidized antibody or antigen-binding fragment thereof may have1, 2, 3, or up to 4 N-terminal 2-hydroxyethylamine moieties oxidized toaldehyde group(s), for linking covalently to a cytotoxic agent describedherein. The N-terminal 2-hydroxyethylamine moiety may be part of aserine, threonine, hydroxylysine, 4-hydroxyornithine or2,4-diamino-5-hydroxy valeric acid residue, preferably Ser or Thr. Forsimplicity, the description below, including the oxidation reaction andany subsequent conjugation with linkers or cytotoxic agents, may referto Ser as a specific example of such N-terminal 2-hydroxyethylaminemoieties, but should generally be construed as referring to allN-terminal 2-hydroxyethylamine moieties. The subject antibody orantigen-binding fragment thereof may comprise an immunoglobulin heavychain variable region (HCVR) having the amino acid sequence set forth inSEQ ID NO: 38; and an immunoglobulin light chain variable region (LCVR)having the amino acid sequence set forth in SEQ ID NO: 33, 35, 37, or 41(preferably SEQ ID NO: 35 or 37). The subject antibody orantigen-binding fragment thereof may also comprise an Ig HCVR having theamino acid sequence set forth in SEQ ID NO: 32, 34, 38, 39, or 40(preferably SEQ ID NO: 34); and an Ig LCVR having the amino acidsequence set forth in SEQ ID NO: 37. The subject antibody orantigen-binding fragment thereof may also comprise an Ig heavy chain(HC) region having the amino acid sequence set forth in SEQ ID NO: 53 or56; and an Ig LCVR having the amino acid sequence set forth in SEQ IDNO: 33, 35, 37, or 41 (preferably SEQ ID NO: 35 or 37). The subjectantibody or antigen-binding fragment thereof may also comprise an Ig HCregion having the amino acid sequence set forth in SEQ ID NO: 48, 50,53, 54, 56, 59, or 60 (preferably SEQ ID NO: 53); and an Ig LCVR havingthe amino acid sequence set forth in SEQ ID NO: 37. In certainembodiments, the second residue from the N-terminus of SEQ ID NOs: 34,38, 50, 53, 54, or 56 is Phe, while in certain other embodiments, thesecond residue from the N-terminus of SEQ ID NOs: 34, 38, 50, 53, 54, or56 is Val.

In certain embodiments, the immunoconjugates of the second embodimentcan be prepared by a first method comprising reacting an oxidizedCD123/IL-3Rα-binding agent having an N-terminal aldehyde described inthe first aspect of the invention with a cytotoxic agent having analdehyde reactive group.

In certain embodiments, the immunoconjugates of the second embodimentcan be prepared by a second method comprising reacting an oxidizedCD123/IL-3Rα-binding agent having an N-terminal aldehyde described inthe first aspect of the invention with a linker compound having analdehyde reactive group to form a modified CD123/IL-3Rα-binding agenthaving a linker bound thereto, followed by reacting the modifiedCD123/IL-3Rα-binding agent with a cytotoxic agent.

In certain embodiments, the immunoconjugates of the second embodimentcan be prepared by a third method comprising contacting an oxidizedCD123/IL-3Rα-binding agent having an N-terminal aldehyde described inthe first aspect of the invention with a cytotoxic agent followed byaddition of a linker compound having an aldehyde reactive group.

In certain embodiments, the immunoconjugates of the second embodimentcan be prepared by a fourth method comprising the steps of:

(a) oxidizing a CD123/IL-3Rα-binding agent having a N-terminal2-hydroxyethylamine moiety (e.g., Ser/Thr) with an oxidizing agent toform an oxidized CD123/IL-3Rα-binding agent having a N-terminal aldehydegroup; and

(b) reacting the oxidized CD123/IL-3Rα-binding agent having theN-terminal aldehyde group with a cytotoxic agent having an aldehydereactive group.

In certain embodiments, the immunoconjugates of the second embodimentcan be prepared by a fifth method comprising the steps of:

(a) oxidizing a CD123/IL-3Rα-binding agent having a N-terminal2-hydroxyethylamine moiety (e.g., Ser/Thr) with an oxidizing agent toform an oxidized CD123/IL-3Rα-binding agent having a N-terminal aldehydegroup;

(b) reacting the oxidized CD123/IL-3Rα-binding agent having theN-terminal aldehyde group with a linker compound having an aldehydereactive group to form a modified CD123/IL-3Rα-binding agent having alinker bound thereto, followed by reacting the modifiedCD123/IL-3Rα-binding agent with a cytotoxic agent.

In certain embodiments, the immunoconjugates of the second embodimentcan be prepared by a sixth method comprising the steps of:

(a) oxidizing the CD123/IL-3Rα-binding agent having a N-terminal2-hydroxyethylamine moiety (e.g., Ser/Thr) with an oxidizing agent toform an oxidized CD123/IL-3Rα-binding agent having a N-terminal aldehydegroup;

(b) contacting the oxidized CD123/IL-3Rα-binding agent having theN-terminal aldehyde group with a cytotoxic agent followed by addition ofa linker compound having an aldehyde reactive group.

In one embodiment, for the first or fourth method described above, thecytotoxic agent having an aldehyde reactive group is represented by thefollowing formula:

-   -   or a pharmaceutically acceptable salt thereof, wherein J_(CB) is        represented by the following formula:

-   -   and the remaining variables are as described above in any one of        1^(st) to 7^(th) and 25^(th) specific embodiments and any more        specific embodiments described therein.

In another embodiment, for the first or fourth method described above,the cytotoxic agent having an aldehyde reactive group is represented bythe following formula:

-   -   or a pharmaceutically acceptable salt thereof, wherein J_(CB) is        as described above and the remaining variables are as described        in the any one of the 19^(th) to 25^(th) specific embodiments        and any more specific embodiments described therein.

In one embodiment, for the second, third, fifth or sixth methoddescribed above, the linker compound is represented by the followingformula:

-   -   wherein J_(D)-SH, —SSR^(d), or —SC(═O)R^(g); R^(d) is phenyl,        nitrophenyl, dinitrophenyl, carboxynitrophenyl, pyridyl or        nitropyridyl; R^(g) is an alkyl; J_(CB) is as described above;        the cytotoxic agent is represented by the following formula:

-   -   and the remaining variables are as described above in any one of        the 8^(th) to 13^(th) and 25^(th) specific embodiments and any        more specific embodiments described therein.

In another embodiment, for the second, third, fifth or sixth methoddescribed above, the linker compound is represented by formula (L^(s)a)above; the cytotoxic compound is represented by the following formula:

-   -   and the remaining variables are as described in any one of the        14^(th) to 18^(th) and 25^(th) specific embodiments and any more        specific embodiments described therein.

In one embodiment, for the first or fourth methods described above, thecytotoxic agent is reacted with an imine-reactive reagent, such asNaHSO₃, to form a modified cytotoxic agent before reacting with theoxidized CD123/IL-3Rα-binding agent having the N-terminal aldehyde. Inone embodiment, the modified cytotoxic agent is not purified beforereacting with the oxidized CBA having the N-terminal aldehyde.Alternatively, the modified cytotoxic agent is purified before reactingwith the oxidized CBA having the N-terminal aldehyde.

In another embodiment, for the second or fifth method described above,the cytotoxic agent is reacted with an imine-reactive reagent, such asNaHSO₃, to form a modified cytotoxic agent before reacting with themodified CD123/IL-3Rα-binding agent having a linker bound thereto. Inone embodiment, the modified cytotoxic agent is purified before reactingwith the modified CD123/IL-3Rα-binding agent having a linker boundthereto. Alternatively, the modified cytotoxic agent is not purifiedbefore reacting with the oxidized CD123/IL-3Rα-binding agent having theN-terminal aldehyde.

In yet another embodiment, for the third or sixth methods describedabove, the reaction of the oxidized CD123/IL-3Rα-binding agent, thecytotoxic agent and the linker compound is carried out in the presenceof an imine reactive reagent, such as NaHSO₃.

Any suitable oxidizing agent can be used in step (a) of the fourth,fifth or sixth method described above. In certain embodiments, theoxidizing agent is a periodate. More specifically, the oxidizing agentis sodium periodate.

Excess molar equivalents of the oxidizing agent relative to theCD123/IL-3Rα-binding agent can be used. In certain embodiments, about2-100, 5-80, 10-50, 1-10 or 5-10 molar equivalents of the oxidizingagent can be used. In certain embodiments, about 10 or about 50equivalents of the oxidizing agent can be used. When large amount of theoxidizing agent is used, short reaction time is used to avoidover-oxidation. For example, when 50 equivalents of the oxidizing agentis used, the oxidation reaction is carried out for about 5 to about 60minutes. Alternatively, when 10 equivalents of the oxidizing agent isused, the reaction is carried out for about 30 minutes to about 24hours. In one embodiment, 5-10 molar equivalents of the oxidizing agentis used and the oxidation reaction is carried out for about 5 to about60 minutes (e.g., about 10 to about 30 minutes, about 20 to about 30minutes).

In certain embodiments, a catalyst is present in the reaction in thefirst, second or third method described above or in the reaction of step(b) in the fourth, fifth or sixth method described above. Any suitablecatalyst in the art can be used. In one embodiment, the catalyst is ananiline or substituted aniline. Exemplary aniline catalyst include, butare not limited to, aniline, o-phenylenediamine, m-phenylenediamine,3,5-diaminobenzoic acid, p-phenylenediamine,2-methyl-p-phenylenediamine, N-methyl-p-phenylenediamine, o-aminophenol,m-aminophenol, p-aminophenol, p-methoxyaniline, 5-methoxy-anthranilicacid, o-aminobenzoid acid, and 4-aminophenethylalcohol. In oneembodiment, the catalyst is 4-aminophenethylalcohol. In certainembodiments, the reaction of step (b) is carried out at pH about 5.0 toabout 6.5. In certain embodiments, the reaction of step (b) is carriedout at pH about 5.0.

In certain embodiments, for the reaction in the first, second or thirdmethod described above or in the reaction of step (b) in the fourth,fifth or sixth method described above, the compound having an aldehydereactive group (e.g., cytotoxic agent, or the linker compound describedherein) is used in molar excess relative to the oxidized cell-bindingagent (e.g., oxidized antibody or oxidized antigen binding portion). Incertain embodiments, the ratio for the compound having an aldehydereactive group to the oxidized cell-binding agent is between about 10:1to about 1.1:1, between about 5:1 to about 2:1. In one embodiment, theratio is about 4:1.

In a third embodiment, the immunoconjugates of the present inventioncomprises a CD123/IL-3Rα-binding agent (including antibody,antigen-binding fragment thereof, or polypeptide comprising the antibodyor antigen-binding fragment thereof) described in the first aspect ofthe invention covalently linked to a cytotoxic agent described hereinthrough the thiol group (—SH) of one or more cysteine residues locatedon the CD123-binding agent.

In a 1^(st) specific embodiment, the immunoconjugate of the thirdembodiment is represented by the following formula:CBA

Cy^(C1))_(W) _(C)   (C1),wherein:

CBA is a CD123/IL-3Rα-binding agent described in the first aspect of theinvention (e.g. a subject antibody or antigen-binding fragment thereofdescribed herein above, or a subject polypeptide thereof describedabove), covalently linked to Cy^(C1) through a cysteine residue;

W_(C) is 1 or 2;

Cy^(C1) is represented by the following formula:

or a pharmaceutically acceptable salt thereof, wherein:

the double line

between N and C represents a single bond or a double bond, provided thatwhen it is a double bond, X is absent and Y is —H or a (C₁-C₄)alkyl; andwhen it is a single bond, X is —H or an amine protecting moiety, Y is—OH or —SO₃M, and M is H⁺ or a cation;

R₅ is —H or a (C₁-C₃)alkyl;

P is an amino acid residue or a peptide containing 2 to 20 amino acidresidues;

R_(a) and R_(b), for each occurrence, are independently —H,(C₁-C₃)alkyl, or a charged substituent or an ionizable group Q;

W′ is —NR^(e′),

R^(e′) is —(CH₂—CH₂—O)_(n)—R^(k);

n is an integer from 2 to 6;

R^(k) is —H or -Me;

R^(x3) is a (C₁-C₆)alkyl; and,

L_(C) is represented by:

wherein s1 is the site covalently linked to CBA, and s2 is the sitecovalently linked to the —C(═O)— group on Cy^(C1); wherein:

R₁₉ and R₂₀, for each occurrence, are independently —H or a(C₁-C₃)alkyl;

m″ is an integer between 1 and 10; and

R^(h) is —H or a (C₁-C₃)alkyl.

In a 2^(nd) specific embodiment, for immunoconjugate of formula (C1),Cy^(C1) is represented by formula (C1a) or (C1a1); and the remainingvariables are as described above in the 1^(st) specific embodiment.

In a 3^(rd) specific embodiment, for immunoconjugate of formula (C1),Cy^(C1) is represented by formula (C1b) or (C1b1); and the remainingvariables are as described above in the 1^(st) specific embodiment.

In a 4^(th) specific embodiment, for immunoconjugate of formula (C1),Cy^(C1) is represented by formula (C1a) or (C1a1); R_(a) and R_(b) areboth H; and R₅ is H or Me; and the remaining variables are as describedabove in the 1^(st) or 2^(nd) specific embodiment.

In a 5^(th) specific embodiment, for immunoconjugate of formula (C1), Pis a peptide containing 2 to 5 amino acid residues; and the remainingvariables are as described above in the 1^(st), 2^(nd) or 4^(th)specific embodiment. In a more specific embodiment, P is selected fromGly-Gly-Gly, Ala-Val, Val-Ala, Val-Cit, Val-Lys, Phe-Lys, Lys-Lys,Ala-Lys, Phe-Cit, Leu-Cit, Ile-Cit, Trp, Cit, Phe-Ala, Phe-N⁹-tosyl-Arg,Phe-N⁹-nitro-Arg, Phe-Phe-Lys, D-Phe-Phe-Lys, Gly-Phe-Lys, Leu-Ala-Leu,Ile-Ala-Leu, Val-Ala-Val, Ala-Leu-Ala-Leu (SEQ ID NO: 55),β-Ala-Leu-Ala-Leu (SEQ ID NO: 57), Gly-Phe-Leu-Gly (SEQ ID NO: 73),Val-Arg, Arg-Val, Arg-Arg, Val-D-Cit, Val-D-Lys, Val-D-Arg, D-Val-Cit,D-Val-Lys, D-Val-Arg, D-Val-D-Cit, D-Val-D-Lys, D-Val-D-Arg,D-Arg-D-Arg, Ala-Ala, Ala-D-Ala, D-Ala-Ala, D-Ala-D-Ala, Ala-Met, andMet-Ala. In another more specific embodiment, P is Gly-Gly-Gly, Ala-Val,Ala-Ala, Ala-D-Ala, D-Ala-Ala, or D-Ala-D-Ala.

In a 6^(th) specific embodiment, for immunoconjugates of formula (C1), Qis —SO₃M; and the remaining variables are as describe above in the1^(st), 2^(nd), 4^(th) or 5^(th) specific embodiment or any morespecific embodiments described therein.

In a 7^(th) specific embodiment, for immunoconjugates of formula (C1),R₁₉ and R₂₀ are both H; and m″ is an integer from 1 to 6; and theremaining variables are as described above in the 1^(st), 2^(nd),3^(rd), 4^(th), 5^(th) or 6^(th) specific embodiment or any morespecific embodiments described therein.

In a 8^(th) specific embodiment, for immunoconjugates of formula (C1),-L-L_(C)- is represented by the following formula:

and the remaining variables are as described above in the 1^(st),2^(nd), 3^(rd), 4^(th), 5^(th), 6^(th) or 7^(th) specific embodiment orany more specific embodiments described therein.

In a 9^(th) specific embodiment, the immunoconjugate of the thirdembodiment is represented by the following formula:

or a pharmaceutically acceptable salt thereof, wherein the double line

between N and C represents a single bond or a double bond, provided thatwhen it is a double bond, X is absent and Y is —H, and when it is asingle bond, X is —H, and Y is —OH or —SO₃M. In a more specificembodiment, the double line

between N and C represents a double bond, X is absent and Y is —H. Inanother more specific embodiment, the double line

between N and C represents a single bond, X is —H and Y is —SO₃M.

In a 10^(th) specific embodiment, the immunoconjugate of the thirdembodiment is represented by the following formula:CBA

Cy^(C2))_(W) _(C)   (C2)wherein:

CBA is a CD123/IL-3Rα-binding agent described in the first aspect of theinvention (e.g., a subject antibody or antigen-binding fragment thereofdescribed herein above, or a subject polypeptide thereof describedabove), covalently linked to Cy^(C2) through a cysteine residue;

W_(C) is 1 or 2;

Cy^(C2) is represented by the following formula:

or a pharmaceutically acceptable salt thereof, wherein:

the double line

between N and C represents a single bond or a double bond, provided thatwhen it is a double bond, X is absent and Y is —H or a (C₁-C₄)alkyl; andwhen it is a single bond, X is —H or an amine protecting moiety, Y is—OH or —SO₃M, and M is H⁺ or a cation;

R^(x1) is a (C₁-C₆)alkyl;

R^(e) is —H or a (C₁-C₆)alkyl;

W′ is —NR^(e′);

R^(e′) is —(CH₂—CH₂—O)_(n)—R^(k);

n is an integer from 2 to 6;

R^(k) is —H or -Me;

R^(x2) is a (C₁-C₆)alkyl;

L_(C)′ is represented by the following formula:

wherein:

s1 is the site covalently linked to the CBA and s2 is the sitecovalently linked to —S— group on Cy^(C2);

Z is —C(═O)—NR₉—, or —NR₉—C(═O)—;

Q is —H, a charged substituent, or an ionizable group;

R₉, R₁₀, R₁₁, R₁₂, R₁₃, R₁₉, R₂₀, R₂₁ and R₂₂, for each occurrence, areindependently —H or a (C₁-C₃)alkyl;

q and r, for each occurrence, are independently an integer between 0 and10;

m and n are each independently an integer between 0 and 10;

R^(h) is —H or a (C₁-C₃)alkyl; and

P′ is an amino acid residue or a peptide containing 2 to 20 amino acidresidues.

In a more specific embodiment, q and r are each independently an integerbetween 1 to 6, more specifically, an integer between 1 to 3. Even morespecifically, R₁₀, R₁₁, R₁₂ and R₁₃ are all H.

In another more specific embodiment, m and n are each independently aninteger between 1 and 6, more specifically, an integer between 1 to 3.Even more specifically, R₁₉, R₂₀, R₂₁ and R₂₂ are all H.

In a 11^(th) specific embodiment, for immunoconjugates of formula (C2),Cy^(C2) is represented by formula (C2a) or (C2a1); and the remainingvariables are as described above in the 10^(th) specific embodiment orany more specific embodiments described therein.

In a 12^(th) specific embodiment, for immunoconjugates of formula (C2),Cy^(C2) is represented by formula (C2b) or (C2b1); and the remainingvariables are as described above in the 10^(th) specific embodiment.

In a 13^(th) specific embodiment, for immunoconjugates of formula (C2),P′ is a peptide containing 2 to 5 amino acid residues; and the remainingvariables are as described in the 10^(th), 11^(th) or 12^(th) specificembodiment or any more specific embodiments described therein. In a morespecific embodiment, P′ is selected from Gly-Gly-Gly, Ala-Val, Val-Ala,Val-Cit, Val-Lys, Phe-Lys, Lys-Lys, Ala-Lys, Phe-Cit, Leu-Cit, Ile-Cit,Trp, Cit, Phe-Ala, Phe-N⁹-tosyl-Arg, Phe-N⁹-nitro-Arg, Phe-Phe-Lys,D-Phe-Phe-Lys, Gly-Phe-Lys, Leu-Ala-Leu, Ile-Ala-Leu, Val-Ala-Val,Ala-Leu-Ala-Leu (SEQ ID NO: 55), β-Ala-Leu-Ala-Leu (SEQ ID NO: 57),Gly-Phe-Leu-Gly (SEQ ID NO: 73), Val-Arg, Arg-Val, Arg-Arg, Val-D-Cit,Val-D-Lys, Val-D-Arg, D-Val-Cit, D-Val-Lys, D-Val-Arg, D-Val-D-Cit,D-Val-D-Lys, D-Val-D-Arg, D-Arg-D-Arg, Ala-Ala, Ala-D-Ala, D-Ala-Ala,D-Ala-D-Ala, Ala-Met, and Met-Ala. In another more specific embodiment,P′ is Gly-Gly-Gly, Ala-Val, Ala-Ala, Ala-D-Ala, D-Ala-Ala, orD-Ala-D-Ala.

In a 14^(th) specific embodiment, for immunoconjugates of formula (C2),-L_(C)′- is represented by the following formula:

In a 15^(th) specific embodiment, for immunoconjugates of (C2), R^(e) isH or Me; R^(x1) is —(CH₂)_(p)—(CR^(f)R^(g))—, and R^(x2) is—(CH₂)_(p)—(CR^(f)R^(g))—, wherein R^(f) and R^(g) are eachindependently —H or a (C₁-C₄)alkyl; and p is 0, 1, 2 or 3; and theremaining variables are as described above in the 10^(th), 11^(th),12^(th), 13^(th), or 14^(th) specific embodiment. More specifically,R^(f) and R^(g) are the same or different, and are selected from —H and-Me.

In a 16^(th) specific embodiment, the immunoconjugate of the thirdembodiment is represented by the following formula:

or a pharmaceutically acceptable salt thereof, wherein the double line

between N and C represents a single bond or a double bond, provided thatwhen it is a double bond, X is absent and Y is —H, and when it is asingle bond, X is —H, and Y is —OH or —SO₃M. In a more specificembodiment, the double line

between N and C represents a double bond, X is absent and Y is —H. Inanother specific embodiment, the double line

between N and C represents a single bond, X is —H and Y is —SO₃M.

In a 17^(th) specific embodiment, the immunoconjugate of the thirdembodiment is represented by the following formula:

wherein:

CBA is a CD123/IL-3Rα-binding agent described in the first aspect of theinvention (e.g., a subject antibody or antigen-binding fragment thereofdescribed herein above, or a subject polypeptide thereof describedabove), covalently linked to Cy^(C3) through a cysteine residue;

W_(C) is 1 or 2;

Cy^(C3) is represented by the following formula:

wherein:

m′ is 1 or 2;

R₁ and R₂, are each independently —H or a (C₁-C₃)alkyl;

L_(C)′ is represented by the following formula:

wherein:

s1 is the site covalently linked to the CBA and s2 is the sitecovalently linked to —S— group on Cy^(C3);

Z is —C(═O)—NR₉—, or —NR₉—C(═O)—;

Q is H, a charged substituent, or an ionizable group;

R₉, R₁₀, R₁₁, R₁₂, R₁₃, R₁₉, R₂₀, R₂₁ and R₂₂, for each occurrence, areindependently —H or a (C₁-C₃)alkyl;

q and r, for each occurrence, are independently an integer between 0 and10;

m and n are each independently an integer between 0 and 10;

R^(h) is —H or a (C₁-C₃)alkyl; and

P′ is an amino acid residue or a peptide containing 2 to 20 amino acidresidues.

In a more specific embodiment, q and r are each independently an integerbetween 1 to 6, more specifically, an integer from 1 to 3. Even morespecifically, R₁₀, R₁₁, R₁₂ and R₁₃ are all H.

In another more specific embodiment, m and n are each independently aninteger between 1 and 6, more specifically, an integer from 1 to 3. Evenmore specifically, R₁₉, R₂₀, R₂₁ and R₂₂ are all H.

In a 18^(th) specific embodiment, for immunoconjugates of formula (C3),P′ is a peptide containing 2 to 5 amino acid residues; and the remainingvariables are as described above in the 17^(th) specific embodiment orany more specific embodiments described therein. In a more specificembodiment, P′ is selected from Gly-Gly-Gly, Ala-Val, Val-Ala, Val-Cit,Val-Lys, Phe-Lys, Lys-Lys, Ala-Lys, Phe-Cit, Leu-Cit, Ile-Cit, Trp, Cit,Phe-Ala, Phe-N⁹-tosyl-Arg, Phe-N⁹-nitro-Arg, Phe-Phe-Lys, D-Phe-Phe-Lys,Gly-Phe-Lys, Leu-Ala-Leu, Ile-Ala-Leu, Val-Ala-Val, Ala-Leu-Ala-Leu (SEQID NO: 55), β-Ala-Leu-Ala-Leu (SEQ ID NO: 57), Gly-Phe-Leu-Gly (SEQ IDNO: 73), Val-Arg, Arg-Val, Arg-Arg, Val-D-Cit, Val-D-Lys, Val-D-Arg,D-Val-Cit, D-Val-Lys, D-Val-Arg, D-Val-D-Cit, D-Val-D-Lys, D-Val-D-Arg,D-Arg-D-Arg, Ala-Ala, Ala-D-Ala, D-Ala-Ala, D-Ala-D-Ala, Ala-Met, andMet-Ala. In another more specific embodiment, P′ is Gly-Gly-Gly,Ala-Val, Ala-Ala, Ala-D-Ala, D-Ala-Ala, or D-Ala-D-Ala.

In a 19^(th) specific embodiment, for immunoconjugates of formula (C3),-L_(C)′- is represented by the following formula:

-   -   wherein M is H⁺ or a cation; and the remaining variables are as        described above in the 17^(th) or 18^(th) specific embodiment or        any more specific embodiments described therein.

In a 20^(th) specific embodiment, for immunoconjugates of formula (C3),m′ is 1 and R₁ and R₂ are both H; and the remaining variables are asdescribed above in the 17^(th), 18^(th) or 19^(th) specific embodimentor any more specific embodiments described therein.

In a 21^(st) specific embodiment, for immunoconjugates of formula (C3),m′ is 2 and R₁ and R₂ are both Me; and the remaining variables are asdescribed above in the 17^(th), 18^(th) or 19^(th) specific embodimentor any more specific embodiments described therein.

In a 22^(nd) specific embodiment, the immunoconjugate of the thirdembodiment is represented by the following formula:

-   -   or a pharmaceutically acceptable salt thereof, wherein DM is a        drug moiety represented by the following formula:

In a 23^(rd) specific embodiment, for the immunoconjugates of the thirdembodiment, M is H⁺, Na⁺ or K⁺; and the remaining variables are asdescribed in any one of the 1^(st) to 22^(nd) specific embodiments orany more specific embodiments described therein.

In any of the above 1^(st) to the 23^(rd) specific embodiments, thesubject antibody or antigen-binding fragment thereof, or polypeptidecomprising the subject antibody or antigen-binding fragment thereof, hasa Cys residue at a location corresponding to the engineered Cys in theheavy chain CH3 domain (i.e., the 5^(th) to the last residue) of SEQ IDNOs: 54 or 56. The subject antibody or antigen-binding fragment thereofmay comprise an immunoglobulin heavy chain region (HC) having the aminoacid sequence set forth in SEQ ID NO: 54; and an immunoglobulin lightchain variable region (LCVR) having the amino acid sequence set forth inSEQ ID NO: 33, 35, 37, or 41 (preferably SEQ ID NO: 35 or 37). Thesubject antibody or antigen-binding fragment thereof may also comprisean Ig heavy chain region having the amino acid sequence set forth in SEQID NO: 56; and an Ig LCVR having the amino acid sequence set forth inSEQ ID NO: 33, 35, 37, or 41 (preferably SEQ ID NO: 35 or 37). Incertain embodiments, the second residue from the N-terminus of SEQ IDNOs: 54 and 56 is Phe, while in certain other embodiments, the secondresidue from the N-terminus of SEQ ID NOs: 54 and 56 is Val.

The immunoconjugates of the third embodiment described above (e.g.,immunoconjugates of any one of the 1^(st) to 23^(rd) specificembodiments or any more specific embodiments described therein) can beprepared by reacting the CBA having one or more free cysteine with acytotoxic agent having a thiol-reactive group described herein.

In one embodiment, the cytotoxic agent having a thiol-reactive group isrepresented by the following formula:

-   -   or a pharmaceutically acceptable salt thereof, wherein -L_(C)        ^(c) is represented by the following formula:

-   -   wherein the variables are as described above in any one of the        1^(st) to 9^(th) and 23^(rd) specific embodiments or any more        specific embodiments described therein.

In another embodiment, the cytotoxic agent having a thiol-reactive groupis represented by the following formula:

-   -   or a pharmaceutically acceptable salt thereof, wherein L_(C)        ^(c) is represented by the following formula:

-   -   wherein the variables are as described above in any one of the        10^(th) to 16^(th) and 23^(rd) specific embodiment or any more        specific embodiments described therein.

In yet another embodiment, the cytotoxic agent having a thiol-reactivegroup is represented by the following formula:

-   -   or a pharmaceutically acceptable salt thereof, wherein L_(C)        ^(c) is described above and the remaining variables are as        described above in any one of the 17^(th) to 23^(rd) specific        embodiments or any more specific embodiments described therein.

In certain embodiments, organic solvents are used in the reaction of theCBA and the cytotoxic agent to solubilize the cytotoxic agent. Exemplaryorganic solvents include, but are not limited to, dimethylacetamide(DMA), propylene glycol, etc. In one embodiment, the reaction of the CBAand the cytotoxic agent is carried out in the presence of DMA andpropylene glycol.

4. Compositions and Methods of Use

The present invention includes a composition (e.g., a pharmaceuticalcomposition) comprising the subject antibodies or antigen-bindingfragments thereof, or immuno-conjugates thereof (e.g., conjugates ofFormulas (L1), (L2), (L3), (S1), (S2), (S3), (S4), (C1), (C2), and (C3))described herein, and a carrier (a pharmaceutically acceptable carrier).The present invention also includes a composition (e.g., apharmaceutical composition) comprising the subject antibodies orantigen-binding fragments thereof, or conjugate of Formulas (L1), (L2),(L3), (S1), (S2), (S3), (S4), (C1), (C2), and (C3)), and a carrier (apharmaceutically acceptable carrier), and further comprising a secondtherapeutic agent. The present compositions are useful for inhibitingabnormal cell growth or treating a proliferative disorder in a mammal(e.g., human), including hematologic cancer, leukemia, or lymphoma.

In particular, the present invention provides pharmaceuticalcompositions comprising one or more of the CD123-binding agents orimmuno-conjugates thereof described herein. In certain embodiments, thepharmaceutical compositions further comprise a pharmaceuticallyacceptable vehicle. These pharmaceutical compositions find use ininhibiting tumor growth and treating cancer in human patients, includinghematologic cancer, leukemia, or lymphoma.

In certain embodiments, formulations are prepared for storage and use bycombining a purified antibody, or immuno-conjugate thereof of thepresent invention with a pharmaceutically acceptable vehicle (e.g.carrier, excipient) (Remington, The Science and Practice of Pharmacy20th Edition Mack Publishing, 2000). Suitable pharmaceuticallyacceptable vehicles include, but are not limited to, nontoxic bufferssuch as phosphate, citrate, and other organic acids; salts such assodium chloride; antioxidants including ascorbic acid and methionine;preservatives (e.g., octadecyldimethylbenzyl ammonium chloride;hexamethonium chloride; benzalkonium chloride; benzethonium chloride;phenol, butyl or benzyl alcohol; alkyl parabens, such as methyl orpropyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; andm-cresol); low molecular weight polypeptides (e.g., less than about 10amino acid residues); proteins such as serum albumin, gelatin, orimmunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;amino acids such as glycine, glutamine, asparagine, histidine, arginine,or lysine; carbohydrates such as monosaccharides, disaccharides,glucose, mannose, or dextrins; chelating agents such as EDTA; sugarssuch as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g., Zn-proteincomplexes); and non-ionic surfactants such as TWEEN or polyethyleneglycol (PEG).

The pharmaceutical compositions described herein can be administered inany number of ways for either local or systemic treatment.Administration can be topical (such as to mucous membranes includingvaginal and rectal delivery) such as transdermal patches, ointments,lotions, creams, gels, drops, suppositories, sprays, liquids andpowders; pulmonary (e.g., by inhalation or insufflation of powders oraerosols, including by nebulizer; intratracheal, intranasal, epidermaland transdermal); oral; or parenteral including intravenous,intraarterial, subcutaneous, intraperitoneal or intramuscular injectionor infusion; or intracranial (e.g., intrathecal or intraventricular)administration. In some particular embodiments, the administration isintravenous. The pharmaceutical compositions described herein can alsobe used in vitro or in ex vivo.

An antibody or immunoconjugate of the invention can be combined in apharmaceutical combination formulation, or dosing regimen as combinationtherapy, with a second compound, such as one that is known to beeffective in treating a disease or disorder of interest. In someembodiments, the second compound is a anti-cancer agent. In someembodiments, the methods encompass administration of the second compoundand an immunoconjugate of the invention that results in a betterefficacy as compared to administration of the immunoconjugate alone. Thesecond compound can be administered via any number of ways, includingfor example, topical, pulmonary, oral, parenteral, or intracranialadministration. In some embodiments, the administration is oral. In someembodiments, the administration is intravenous. In some embodiments, theadministration is both oral and intravenous.

An antibody or immunoconjugate can also be combined in a pharmaceuticalcombination formulation, or dosing regimen as combination therapy, withan analgesic, or other medications.

An antibody or immunoconjugate can be combined in a pharmaceuticalcombination formulation, or dosing regimen as combination therapy, witha second compound having anti-cancer properties. The second compound ofthe pharmaceutical combination formulation or dosing regimen can havecomplementary activities to the ADC of the combination such that they donot adversely affect each other. Pharmaceutical compositions comprisingthe CD123-binding agent and the second anti-cancer agent are alsoprovided.

The present invention includes a method of inhibiting abnormal cellgrowth or treating a proliferative disorder in a mammal (e.g., human)comprising administering to said mammal a therapeutically effectiveamount of the subject antibodies or antigen-binding fragments thereof,or immuno-conjugates (e.g., conjugates of formulas (L1), (L2), (L3),(S1), (S2), (S3), (S4), (C1), (C2), and (C3)) described herein, or acomposition thereof, alone or in combination with a second therapeuticagent.

In certain embodiments, the abnormal cell growth or proliferativedisorder in a mammal is a disease or condition associated with orcharacterized by the expression of CD123, such as cancer, includinghematologic cancer, leukemia, or lymphoma. In certain embodiments, theproliferative disorder is a cancer of a lymphatic organ, or ahematological malignancy.

For example, the cancer may be selected from the group consisting of:acute myeloid leukemia (AML, including CD33-low AML, P-glycoproteinpositive AML, relapsed AML, or refractory AML), chronic myelogenousleukemia (CML), including blastic crisis of CML and Abelson oncogeneassociated with CML (Bcr-ABL translocation), myelodysplastic syndrome(MDS), acute B lymphoblastic leukemia or B-cell acute lymphoblasticleukemia (B-ALL), chronic lymphocytic leukemia (CLL), includingRichter's syndrome or Richter's transformation of CLL, hairy cellleukemia (HCL), acute promyelocytic leukemia (APL), B-cell chroniclymphoproliferative disease (B-CLPD), atypical chronic lymphocyticleukemia (preferably with a marked CD11c expression), blasticplasmacytoid dendritic cell neoplasm (BPDCN), non-Hodgkin lymphomas(NHL), including mantel cell leukemia (MCL), and small lymphocyticlymphoma (SLL), Hodgkin's lymphoma, systemic mastocytosis, and Burkitt'slymphoma.

In certain embodiments, the B-ALL is a CD19 positive B-ALL. In certainother embodiments, the B-ALL is a CD19 negative B-ALL.

In certain embodiments, the cancer has at least one negative prognosticfactor, e.g., overexpression of P-glycoprotein, overexpression of EVI1,a p53 alteration, DNMT3A mutation, FLT3 internal tandem duplication.

In certain embodiments, the therapeutically effective amount of thesubject antibodies or antigen-binding fragments thereof, orimmuno-conjugates (e.g., conjugates of formulas (L1), (L2), (L3), (S1),(S2), (S3), (S4), (C1), (C2), and (C3)) described herein, or acomposition thereof, alone or in combination with a second therapeuticagent, preferentially inhibits the proliferation of leukemic stem cells(LSCs), leukemia progenitors (LPs), and/or leukemic blasts, over normalhematopoietic stem cells (HSCs). In certain embodiments, IC₅₀ value orthe half maximum concentration of the above subject agents to inhibitthe proliferation of leukemic stem cells (LSCs), leukemia progenitors(LPs), and/or leukemic blasts, is at least 10-, 20-, 30-, 40-, 50-, 60-,70-, 80-, 90-, 100-, 150-, 300-, 500-fold or more lower than that forthe normal hematopoietic stem cells (HSCs).

In certain embodiments, an anti-leukemia therapy of the invention notonly targets and kills leukemic blasts, but preferably also targets andkills leukemic progenitors (LP) and leukemic stem sells (LSCs). Incertain embodiments, the therapy is also less selective against normalHSCs. In certain embodiments, CD123 expression on LSCs, LPs and leukemiablasts are much higher (e.g., at least 20-25 fold higher in LSC, AMLprogenitors, and AML blasts) as compared to normal lymphocytes (whichmay be close to negative). In certain embodiments, CD123 expressionlevels on LPs and LSCs are at least as high as those on leukemic blasts.

Similarly, the present invention provides a method for inducing celldeath in selected cell populations comprising contacting target cells ortissue containing target cells with an effective amount of the subjectantibodies or antigen-binding fragments thereof, or immuno-conjugates ofthe present invention. The target cells are cells to which thecell-binding agent of the conjugates can bind.

If desired, other active agents, such as other anti-tumor agents, may beadministered along with the conjugate.

Cancer therapies and their dosages, routes of administration andrecommended usage are known in the art and have been described in suchliterature as the Physician's Desk Reference (PDR). The PDR disclosesdosages of the agents that have been used in treatment of variouscancers. The dosing regimen and dosages of these aforementionedchemotherapeutic drugs that are therapeutically effective will depend onthe particular cancer being treated, the extent of the disease and otherfactors familiar to the physician of skill in the art and can bedetermined by the physician. The contents of the PDR are expresslyincorporated herein in its entirety by reference. One of skill in theart can review the PDR, using one or more of the following parameters,to determine dosing regimen and dosages of the chemotherapeutic agentsand conjugates that can be used in accordance with the teachings of thisinvention. These parameters include: Comprehensive index; Manufacturer;Products (by company's or trademarked drug name); Category index;Generic/chemical index (non-trademark common drug names); Color imagesof medications; Product information, consistent with FDA labeling;Chemical information; Function/action; Indications & Contraindications;Trial research, side effects, warnings.

Examples of in vitro uses include treatments of autologous bone marrowprior to their transplant into the same patient in order to killdiseased or malignant cells: treatments of bone marrow prior to theirtransplantation in order to kill competent T cells and preventgraft-versus-host-disease (GVHD); treatments of cell cultures in orderto kill all cells except for desired variants that do not express thetarget antigen; or to kill variants that express undesired antigen.

The conditions of non-clinical in vitro use are readily determined byone of ordinary skill in the art.

Examples of clinical ex vivo use are to remove tumor cells or lymphoidcells from bone marrow prior to autologous transplantation in cancertreatment or in treatment of autoimmune disease, or to remove T cellsand other lymphoid cells from autologous or allogenic bone marrow ortissue prior to transplant in order to prevent GVHD. Treatment can becarried out as follows. Bone marrow is harvested from the patient orother individual and then incubated in medium containing serum to whichis added the cytotoxic agent of the invention, concentrations range fromabout 10 μM to 1 pM, for about 30 minutes to about 48 hours at about 37°C. The exact conditions of concentration and time of incubation, i.e.,the dose, are readily determined by one of ordinary skill in the art.After incubation the bone marrow cells are washed with medium containingserum and returned to the patient intravenously according to knownmethods. In circumstances where the patient receives other treatmentsuch as a course of ablative chemotherapy or total-body irradiationbetween the time of harvest of the marrow and reinfusion of the treatedcells, the treated marrow cells are stored frozen in liquid nitrogenusing standard medical equipment.

For clinical in vivo use, the cytotoxic compounds or conjugates of theinvention will be supplied as a solution or a lyophilized powder thatare tested for sterility and for endotoxin levels.

Suitable pharmaceutically acceptable carriers, diluents, and excipientsare well known and can be determined by those of ordinary skill in theart as the clinical situation warrants. Examples of suitable carriers,diluents and/or excipients include: (1) Dulbecco's phosphate bufferedsaline, pH about 7.4, containing or not containing about 1 mg/mL to 25mg/mL human serum albumin, (2) 0.9% saline (0.9% w/v NaCl), and (3) 5%(w/v) dextrose; and may also contain an antioxidant such as tryptamineand a stabilizing agent such as Tween 20.

The method of the invention for inducing cell death in selected cellpopulations, for inhibiting cell growth, and/or for treating cancer, canbe practiced in vitro, in vivo, or ex vivo.

EXAMPLES Example 1 Generation of Mouse Monoclonal Antibodies AgainstHuman and Cynomolgus CD123 Antigen

To produce murine anti-CD123 antibodies, wild type BALB/c female mice(Charles River Laboratory, Wilmington, Mass.) were injectedsubcutaneously with human CD123 expressing stable 300-19 cell line,which is a BALB/c derived pre-B cell line (M. G. Reth et al., 1985,Nature, 317: 353-355), in PBS at dose of 5×10⁶ cells/mouse every 2 weeksfor five times. Three days before being sacrificed for hybridomageneration, the immunized mice received intraperitoneal injection ofanother dose of antigen. The spleen from the mouse was collectedaccording to standard animal protocols and was ground between twosterile, frosted microscopic slides to obtain a single cell suspensionin RPMI-1640 medium. After the red blood cells were lysed with ACKlysing buffer, the spleen cells were then mixed with murine myelomaP3X63Ag8.653 cells (P3 cells) (J. F. Kearney et al., 1979, J. Immunol,123: 1548-1550) at ratio of 1 P3 cells:3 spleen cells. The mixture ofspleen cells and P3 cells was washed and treated with pronase in fusionmedia (0.3 M mannitol/D-sorbitol, 0.1 mM CaCl₂, 0.5 mM MgCl₂ and 1 mg/mLBSA) at room temperature for 3 min. The reaction was stopped by additionof Fetal Bovine Serum (FBS, Invitrogen); cells were then washed,resuspended in 2 mL cold fusion media and fused with BTX ECM 2001electrofusion machine (Harvard Apparatus). The fused cells were addedgently to RPMI-1640 selection medium containinghypoxanthine-aminopterin-thymidine (HAT) (Sigma Aldrich), incubated for20 min at 37° C., and then seeded into flat bottom 96-well plates at 200μL/well. The plates were then incubated in 5% CO₂ incubator at 37° C.until hydridoma clones were ready for antibody screening. Othertechniques of immunization and hybridoma production can also be used,including those described in J. Langone and H. Vunakis (Eds., Methods inEnzymology, Vol. 121, Immunochemical Techniques, Part I, Academic Press,Florida); and E. Harlow and D. Lane (Antibodies: A Laboratory Manual,1988, Cold Spring Harbor Laboratory Press, New York, N.Y.).

Hybridoma Screening and Selection

Hybridoma screening was done using flow cytometric binding assay withhuman CD123 expressing stable 300-19 cell lines and wild-type 300-19cells. In brief, the wild-type 300-19 cells were first labeled withCELLTRACE™ far red DDAO-SE (Invitrogen), mixed with untreated cells at1:1 ratio and incubated with the hybridoma supernatant for 2 hours onice. Cells were then washed, incubated with PE-labeled anti mouse IgG(Jackson Immunoresearch), washed, fixed with formalin and analyzed usingFACS array (BD Bioscience). The hybridoma with specific reactivity tohuman CD123 antigen were expanded and the supernatants were rescreenedby flow cytometric binding assay using three independent cell lines:human CD123 expressing stable 300-19 cell line, cynomolgus CD123expressing stable 300-19 cell line and wild type 300-19 cell line. Thehybridoma with positive binding to human and cynomolgus CD123 antigensbut negative on wild type 300-19 cells were further subcloned bylimiting dilution. One subclone from each hybridoma, which showedspecific binding to human and cynomolgus CD123 antigens, was selectedfor subsequent analysis.

A total of six fusions were conducted over the course of thisinvestigation. Approximately 6,000 hybridomas were screened, 33hybridomas specific for human and cynomolgus CD123 antigens weregenerated and 18 hybridomas were subcloned. Stable subclones werecultured and the isotype of the monoclonal antibody was identified usingcommercial mouse IgG isotyping reagents (such as the IsoStrip MouseMonoclonal Antibody Isotyping Kit by Roche Diagnostics GmbH, Germany,Product No. 11493027001).

Antibody Purification

Antibodies were purified from hybridoma subclone supernatants usingstandard methods, such as, for example Protein A or G chromatography(HiTrap Protein A or G HP, 1 mL, Amersham Biosciences). Briefly,supernatant was prepared for chromatography by the addition of 1/10volume of 1 M Tris/HCl buffer, pH 8.0. The pH-adjusted supernatant wasfiltered through a 0.22 μm filter membrane and loaded onto columnequilibrated with binding buffer (PBS, pH 7.3). The column was washedwith binding buffer until a stable baseline was obtained with noabsorbance at 280 nm. Antibody was eluted with 0.1 M acetic acid buffercontaining 0.15 M NaCl, pH 2.8, using a flow rate of 0.5 mL/min.Fractions of approximately 0.25 mL were collected and neutralized by theaddition of 1/10 volume of 1M Tris/HCl, pH 8.0. The peak fraction(s) wasdialyzed overnight twice against 1×PBS and sterilized by filteringthrough a 0.2 μm filter membrane. Purified antibody was quantified byabsorbance at A280.

Protein A purified fractions were further polished using ion exchangechromatography (IEX) with quaternary ammonium (Q) chromatography formurine antibodies. Briefly, samples from protein A purification werebuffer exchanged into binding buffer (10 mM Tris, 10 mM sodium chloride,pH 8.0) and filtered through 0.22 μm filer. The prepared sample was thenloaded onto a Q fast flow resin (GE Lifesciences) that was equilibratedwith binding buffer at a flow rate of 120 cm/hr. Column size was chosento have sufficient capacity to bind all the MAb in the sample. Thecolumn was then washed with binding buffer until a stable baseline wasobtained with no absorbance at 280 nm. Antibody was eluted by initiatinga gradient from 10 mM to 500 mM sodium chloride in 20 column volume(CV). Peak fractions were collected based on absorbance measurement at280 nm (A280). The percentage of monomer was assessed with sizeexclusion chromatography (SEC) on a TSK gel G3000SWXL, 7.8×300 mm with aSWXL guard column, 6.0×40 mm (Tosoh Bioscience, Montgomeryville, Pa.)using an Agilent HPLC 1100 system (Agilent, Santa Clara, Calif.).Fractions with monomer content above 95% were pooled, buffer exchangedto PBS (pH 7.4) using a TFF system, and sterilized by filtering througha 0.2 μm filter membrane. The IgG concentration of purified antibody wasdetermined by A280 using an extinction coefficient of 1.47. Alternativemethods such as ceramic hydroxyapatite (CHT) were also used to polishantibodies with good selectivity. Type II CHT resin with 40 μm particlesize (Bio-Rad Laboratories) were used with a similar protocol asdescribed for IEX chromatography. The binding buffer for CHT correspondsto 20 mM sodium phosphate, pH 7.0 and antibody was eluted with agradient of 20-160 mM sodium phosphate over 20 CV.

Example 2 Cloning and Sequencing of the V_(L) and V_(H) Regions of theAnti-CD123 Antibodies Cloning of the V_(L) and V_(H) Regions

Total cellular RNA was prepared from 5×10⁶ cells of the CD123 hybridomasusing an RNeasy kit (QIAgen) according to the manufacturer's protocol.cDNA was subsequently synthesized from total RNA using the SuperScriptII cDNA synthesis kit (Invitrogen).

The PCR procedures for amplifying the antibody variable region cDNAsderived from hybridoma cells were based on methods described in Wang etal. ((2000) J Immunol Methods. 233:167-77) and Co et al. ((1992) JImmunol. 148:1149-54). The V_(L) and V_(H) sequences were amplified bydegenerate primers on the 5′-end and either murine kappa or IgG₁constant region specific primers respectively on the 3′-end. Thepurified amplicons were sent to Beckman Coulter Genomics for sequencing.

Since the degenerate primers used to clone the V_(L) and V_(H) cDNAsequences alter the 5′-end, additional sequencing efforts were needed toverify the complete cDNA sequences. The preliminary sequences wereentered into a search query of the NCBI IgBlast site to identify themurine germline sequences from which the antibody sequences had beenderived. PCR primers were then designed to anneal to the germline linkedleader sequence of the murine antibody so that this new PCR reactionwould yield a complete variable region cDNA sequence, unaltered by thePCR primers.

Mass Determination for Sequence Confirmation

The variable regions cDNA sequences obtained for each of the anti-CD123antibodies were combined with germline constant region sequences toobtain full length antibody cDNA sequences. The molecular weights of theheavy and light chains were then calculated from translations of thecDNA sequences and compared with the molecular weights obtained by LC/MSanalyses of the purified murine anti-CD123 antibodies. The observedmolecular weights for each of the light and heavy chains matched theexpected values.

Example 3 Antibody Humanization Recombinant Antibody Expression

The confirmed variable region amino acid sequences for the murine CD123antibodies were codon-optimized, synthesized and cloned in-frame withhuman antibody constant regions by Blue Heron Biotechnology to buildchimeric versions of the CD123 antibodies. The vectors, constantregions, and cloning schemes used for the chimeric CD123 antibodies wereidentical to those used for the humanized CD123 antibodies describedbelow. The chimeric antibody chCD123-6 is comprised of the mousevariable region sequences of SEQ ID NOs: 28 and 29, respectively,together with the human IgG1 and Kappa constant sequences for the heavyand light chains, respectively. The light chain variable region wascloned into the EcoRI and BsiWI sites of the pAbKZeo plasmid and theheavy chain variable region was cloned into the HindIII and Apa1 sitesof the pAbG1Neo plasmid. These expression constructs were transientlyproduced in either adherent HEK-293T cells using suspension adaptedHEK-293T cells using a modified PEI procedure in shake flasks. The PEItransient transfections were performed as previously described (Durocheret al., Nucleic Acids Res. 30(2):E9 (2002)), except the HEK-293T cellswere grown in Freestyle 293 (Invitrogen) and the culture volume was leftundiluted after the addition of the PEI-DNA complexes. The transfectionswere incubated for a week and then the cleared supernatants werepurified by standard Protein A chromatography followed by polishingchromatography procedures.

Antibody Humanization

The murine CD123-6 antibody was humanized using complementarydetermining region (CDR) grafting procedures substantially as describedin Jones et al., Nature 321: 604-608, 1986, Verhoeyen et al., Science239: 1534-1536, 1988, U.S. Pat. No. 5,225,539, and U.S. Pat. No.5,585,089. CDR grafting generally consists of replacing the Fv frameworkregions (FRs) of a mouse antibody with human antibody Fv frameworkregions while preserving the mouse CDR residues critical for thespecific antigen-binding properties of the parent antibody. ExemplaryCDRs of the CD123-6 antibody following the Kabat numbering scheme andthe Kabat CDR definitions are as indicated in Table A below.

TABLE A CD123-6 CDRs (CDR grafting) Light Chain CDR-L1: Murine:KASQDINSYLS (SEQ ID NO: 19) CDR grafted: RASQDINSYLS (SEQ ID NO: 20)CDR-L2: RVNRLVD (SEQ ID NO: 21) CDR-L3: LQYDAFPYT (SEQ ID NO: 22)Heavy Chain CDR-H1: SSIMH (SEQ ID NO: 5) CDR-H2:YIKPYNDGTKYNEKFKG (SEQ ID NO: 8) CDR-H3: EGGNDYYDTMDY (SEQ ID NO: 11)

The CDR-grafting process begins by selecting appropriate human acceptorframeworks, typically those derived from human antibody genes sharingthe highest sequence homology to the parent murine antibody. The humanimmunoglobulin germline light and heavy chain sequences with the highesthomology to the murine CD123-6 antibody was identified utilizing theinteractive tool, DomainGapAlign, of the International ImMunoGeneTicsinformation System® (IMGT (http column double slash imgt dot cines dotfr slash) as described in Ehrenmann et al., Nucleic Acids Res. 38:D301-307 (2010). The human germline sequences selected as the acceptorframeworks for the V_(L) and V_(H) domains of CD123-6 antibody wereIGKV1-16*01 and IGHV1-46*03, respectively.

Sequence alignment among the relevant portion of the original muCD123-6V_(L) sequence, the corresponding human germline sequence IGKV1-16*01,and the corresponding huCD123-6VLGv1 and huCD123-6VLGv4 sequences isshown below.

1                                                          61muCD123-6 VL (1)DIKMTQSPSSMYASLGERVTITCKASQDINSYLSWFQQKPGKSPKTLIYRVNRLVDGVPSRSEQ ID NO: 79huCD123-6VLGV1 (1)--Q-------LS--V-D------R---------A--------A--S---------S-----(SEQ ID NO: 80)huCD123-6VLGV4 (1)--Q-------LS--V-D------R------------------A------------------(SEQ ID NO: 81)IGKV1-16*01 (1)--Q-------LS--V-D------R---G-SN--A--------A--S---AASS-QS-----(SEQ ID NO: 82)62                                          108 muCD123-6 VL (62)FSGSGSGQDYSLTISSLEYEDMGIYYCLQYDAFPYTFGGGTKLEIKR(SEQ ID NO: 83)huCD123-6VLGV1 (62)-------T-FT------QP--FAT--------------Q---V----(SEQ ID NO: 84)huCD123-6VLGV4 (62)-------N--T------QP--FAT--------------Q---V----(SEQ ID NO: 85)IGKV1-16*01 (62)-------T-FT------QP--FAT---Q--NSY-             (SEQ ID NO: 86)

Sequence alignment among the relevant portion of the original muCD123-6V_(H) sequence, the corresponding human germline sequence IGHV1-46*03,and the corresponding huCD123-6VHGv1, huCD123-6VHGv6, and huCD123-6VHGv7sequences is shown below.

1                                                          61muCD123-6 VH (1)EFQLQQSGPELVKPGASVKMSCKASGYIFTSSIMHWMKQKPGQGLEWIGYIKPYNDGTKYN (SEQ ID NO: 87)huCD123-6VHGV1 (1)QV--V---A-VK-------V-------G--------VR-A-------M------------- (SEQ ID NO: 88)huCD123-6VHGV6 (1)Q---V---A-VK-------V----------------VR-A--------------------- (SEQ ID NO: 89)huCD123-6VHGV7 (1)QV--V---A-VK-------V----------------VR-A--------------------- (SEQ ID NO: 90)IGHV1-46*03 (1)QV--V---A-VK-------V-------T---YY---VR-A-------M-I-N-SGGS-S-A (SEQ ID NO: 91)62                                                       121muCD123-6 VH (62)EKFKGKATLTSDKSSSTANMELNSLTSEDSAVYYCAREGGNDYYDTMDYWGQGTSVTVSS(SEQ ID NO: 92)huCD123-6VHGV1 (62)-----RV-M-R-T-T--VY---S--R---T------------------------L-----(SEQ ID NO: 92)huCD123-6VHGV6 (62)-----R------R-T---Y---S--R---T------------------------L-----(SEQ ID NO: 94)huCD123-6VHGV7 (62)-----R------R-T---Y---S--R---T------------------------L-----(SEQ ID NO: 95)IGHV1-46*03 (62) Q--Q-RV-M-R-T-T--VY---S--R---T------- (SEQ ID NO: 96)

The humanized DNA constructs were synthesized, expressed, and therecombinant antibodies purified as described above for subsequent CD123binding analysis compared with the parent antibody.

It is well established that framework residues can also make structuralcontributions to antigen-binding and may be re-introduced asback-mutations to maximally preserve antigen-binding affinity. Foote andWinter, J. Mol. Biol. 224: 487-499 (1992). A platform of residuesdirectly underneath the CDRs, referred to as vernier zone residues, mayhelp to preserve the conformation of the CDR loops that direct thespecificity and affinity of the antibody. Thus variants containing oneor more back-mutations of the vernier zone residues were made, andsubsequently evaluated for antigen-binding as well as for the functionalIL3 inhibition activity. The vernier zone residue back-mutations testedincluded 3 residues in the V_(L) (position 46 in FW-L2, and positions69, 71 in FW-L3) and 8 residues in the V_(H) (positions 2, 28 in FW-H1,position 48 in FW-H2, and positions 67, 69, 71, 73, 78 in FW-H3).Several CDR-grafted CD123-6 antibodies with vernier zone back-mutationsexhibited IL3 inhibition activity on TF-1 cells as exemplified byversion 4.6 or referred to as “Gv4.6” herein (V_(L) Gv4 and V_(H) Gv6)and version 4.7 or referred to as “Gv4.7” herein (V_(L) Gv4 and V_(H)Gv7) (FIG. 1).

The specific framework residue usage of the CDR-grafted CD123-6antibodies described are given in Tables B and C below.

TABLE B CDR-grafting of CD123-6 antibody V_(L) CD123-6-V_(L) Human HumanKabat Murine (CDR-graft) (CDR-graft) position residue v1 residue v4residue 3 K Q Q 11 M L L 12 Y S S 15 L V V 17 E D D 43 S A A 46 T S T 69Q T N 71 Y F Y 72 S T T 79 E Q Q 80 Y P P 83 M F F 84 G A A 85 I T T 100G Q Q 104 L V V 24 K R R

TABLE C CDR-grafting of CD123-6 antibody V_(H) CD123-6-V_(H) Human HumanHuman (CDR-graft) (CDR-graft) (CDR-graft) Kabat Murine v1 v6 v7 positionresidue residue residue residue  1 E Q Q Q  2 F V F V  5 Q V V V  9 P AA A 11 L V V V 12 V K K K 20 M V V V 28 I G I I 37 M V V V 38 K R R R 40K A A A 48 I M I I 66 K R R R 67 A V A A 69 L M L L 71 S R S S 73 K T RR 75 S T T T 78 A V A A 79 N Y Y Y  82a N S S S 83 T R R R 87 S T T T108  S L L L

Additionally, to minimize concerns about the impact of conjugatinglysines that fall in CDRs, lysine 24 in murine CD123-6 antibody CDR-L1was replaced with arginine in CDR grafting (see Table A above).

The CD123-6 antibody was also humanized by variable domain resurfacing,following methods previously described, Roguska et al., Proc. Natl.Acad. Sci., USA, 91(3):969-973, 1994 and Roguska et al., Protein Eng.9(10):895-904, 1996. Resurfacing generally involves identification ofthe variable region framework surface residues in both the light andheavy chains and replacing them with human equivalents. The murine CDRsand buried framework residues are preserved in the resurfaced antibody.Exemplary CDRs of CD123-6 antibodies are defined as indicated in TableD.

TABLE D CD123-6 antibody CDRs (Resurfacing) Light Chain CDR-Ll: Murine:KASQDINSYLS (SEQ ID NO: 19) Resurfaced: RASQDINSYLS (SEQ ID NO: 20)CDR-L2: RVNRLVD (SEQ ID NO: 21) CDR-L3: LQYDAFPYT (SEQ ID NO: 22)Heavy Chain CDR-H1: SSIMH (SEQ ID NO: 5) CDR-H2: Murine and resurfacedYIKPYNDGTK (SEQ ID NO: 6) v1.1: Resurfaced v1.0:YIRPYNDGTR (SEQ ID NO: 7) CDR-H3: EGGNDYYDTMDY (SEQ ID NO: 11)Kabat CD123-6 CDR-H2 Murine CDR-H2: YIKPYNDGTKYNEKFKG (SEQ ID NO: 8)Resurfaced v1.0 YIRPYNDGTRYNQKFQG (SEQ ID CDR-H2: NO: 9) Resurfaced v1.1YIKPYNDGTKYNQKFQG (SEQ ID CDR-H2: NO: 10) *The double underlinedsequence marks the portion of the Kabat heavy chain CDR-H2 that was notconsidered a CDR for resurfacing.

To minimize concerns about the impact of conjugating lysines that fallwithin CDRs, lysine 24 of the murine CD123-6 antibody light chain CDR-L1was replaced with arginine. Similarly, lysines 52 and 59 of the murineCD123-6 antibody heavy chain CDR-H2 were replaced with arginines forresurfaced version 1.0. The AbM heavy chain CDR-H2 definition wasemployed for resurfacing so the table provides those as well asexemplary Kabat defined heavy chain CDR-H2 sequences for both the murineand human versions of CD123-6 antibody.

Surface residue positions were defined as any position with a relativeaccessibility of 30% or greater (Pedersen et al., J. Mol. Biol. 235:959-973, 1994). The calculated surface residues were then aligned withhuman germline surface sequences to identify the most homologous humansurface sequence. The human germline sequence used as the replacementsurface for the light chain variable domain of CD123-6 antibody wasIGKV1-16*01 while IGHV1-69*10 was used as the replacement surface forheavy chain variable domain. The specific framework surface residuechanges are given in Tables E and F below.

TABLE E Resurfacing of CD123-6 antibody V_(L) CD123-6 V_(L) Human KabatMurine (resurface) position residue residue 1 D D 3 K O 5 T T 9 S S 12 YS 15 L V 18 R R 40 P P 41 G G 57 G G 60 S S 67 S S 80 Y P 81 E E 100 G O103 K K 107 K K 108 R R 24 K R

TABLE F Resurfacing of CD123-6 antibody V_(H) CD123-6 V_(H) Human Human(resurface) Kabat Murine v1.0 v1.1 position residue residue residue  1 EO O  2 F V F  3 Q Q Q  5 Q V V  9 P A A 11 L V V 13 K K K 14 P P P 19 KK K 23 K K K 28 I T T 41 P P P 42 G G G 43 Q Q Q 61 E O O 62 K K K 64 KO O 65 G G G 73 K R K 74 S S S   82B S S S 84 S S S 85 E E E 105  Q Q Q112  S S S 52 K R K 59 K R K

Sequence alignments below show the resurfaced sequences for CD123-6antibody variable domain of both light (V_(L)) and heavy (V_(H)) chainwith their murine counterparts. See SEQ ID NO: 41 for the resurfacedhuCD123-6 V_(L) sequence, and SEQ ID NOs: 39 and 40 for the resurfacedhuCD123-6 V_(H) sequences.

1                                                          61muCD123-6 VLDIKMTQSPSSMYASLGERVTITCKASQDINSYLSWFQQKPGKSPKTLIYRVNRLVDGVPSR(SEQ ID NO: 79)huCD123-6 VL--Q--------S--V--------R-------------------------------------(SEQ ID NO: 74)62                                          108 muCD123-6 VLFSGSGSGQDYSLTISSLEYEDMGIYYVLQYDAFPYTFGGGTKLEIKR(SEQ ID NO: 83)huCD123-6 VL------------------P-------------------Q--------(SEQ ID NO: 75)1                                                          61muCD123-6 VHEFQLQQSGPELVKPGASVKMSCKASGYIFTSSIMHWMKQKPGQGLEWIGYIKPYNDGTKYN(SEQ ID NO: 87)huCD123-6 VHV1.0QV--V---A-V----------------T-----------------------R------R--(SEQ ID NO: 76)huCD123-6 VHV1.1Q---V---A-V----------------T---------------------------------(SEQ ID NO: 77)62                                                       121muCD123-6 VHEKFKGKATLTSDKSSSTANMELNSLTSEDSAVYYCAREGGNDYYDTMDYWGQGTSVTVSS(SEQ ID NO: 92)huCD123-6 VHV1.0Q--Q--------R-----------------------------------------------(SEQ ID NO: 78)huCD123-6 VHV1.1Q--Q--------------------------------------------------------(SEQ ID NO: 97)

Example 4 Screen for Anti-CD123 Antibodies that Inhibit IL3-MediatedSignaling and Proliferation

The ability of anti-CD123 antibodies to inhibit IL3-mediated signalingand proliferation was examined in vitro using the erythroleukemia cellline TF-1 that can proliferate only in the presence of one of thefollowing growth factors, either IL-3 or GM-CSF. TF-1 cells werecultured in the complete RPMI medium (RPMI-1640, 10% fetal bovine serumand 50 μg/mL gentamicin sulfate; all reagents were from Invitrogen)supplemented with GM-CSF (2 ng/mL). Prior to setting up theproliferation assays, the cells were washed and then starved of thegrowth factors overnight. To block Fc receptors on the cell surface, theculture medium was supplemented with 100 nM chKTI antibody (anon-binding antibody of the same isotype). TF-1 cells were plated at6,000 cells per well in the complete RPMI medium in either the presenceor absence of 10 μg/mL of an anti-CD123 antibody. A growth factor,either IL-3 (1 ng/mL) or GM-CSF (2 ng/mL), was added to the cells toinitiate cell proliferation. Cells were incubated at 37° C. in ahumidified 5% CO₂ incubator for 3 days. The relative numbers of viablecells in each well were determined by the colorimetric WST-8 assay(Dojindo Molecular Technologies, Inc., Rockville, Md., US). WST-8 isreduced by dehydrogenases in viable cells to an orange formazan productthat is soluble in tissue culture medium. The amount of formazanproduced is directly proportional to the number of viable cells. WST-8was added into the wells at 10% of the final volume, and the plates wereincubated at 37° C. in a humidified 5% CO₂ incubator for an additional2-6 hours. Then the absorbance was measured on a plate-readerspectrophotometer in the dual wavelength mode 450 nm/650 nm, and theabsorbance at the 650 nm (non-specific light scattering by cells) wassubtracted from that at the 450 nm. The relative cell number in eachwell was calculated by first correcting for the medium backgroundabsorbance, and then dividing the value of each sample treated with anantibody by the average values of wells with the untreated cells(control).

The results from a typical assay are presented in FIGS. 2A and 2B.Consistent with the previously reported data (Sun et al. 1996), the 7G3but not 6H6 or 9F5 antibody substantially inhibited IL-3 dependentproliferation. Unexpectedly, several anti-CD123 antibodies generated inthis study were able to inhibit IL-3 dependent proliferation of TF-1cells even more significantly than 7G3 (FIG. 2A). For example, theantibody 3, 6 and 14 reduced the number of TF-1 cells to less than 5% ofthat in the control (TF-1 cells grown in the absence of an antibody),whereas 7G3 antibody reduced the number of cells to 15% of that in thecontrol. In contrast, the treatment with the other anti-CD123 antibodies(e.g., the antibodies 2, 5, 7, 8, 9, 12, 13, 16, 18, 20, 21 and 22) hadonly a minimal effect on the cell proliferation or no effect at all.

The inhibition of TF-1 cell proliferation by the antibodies 3, 6, 14 and7G3 was IL-3 dependent, as these antibodies had no inhibitory effectwhen the cells were grown in the presence of another growth factorGM-CSF (FIG. 2B).

Next, the concentrations of the antibodies 3, 6, 14 (renamed muCD123-3,muCD123-6, muCD123-14, respectively) and 7G3 that were needed to inhibitIL3-dependent proliferation were determined. TF-1 cells were plated at6,000 cells per well in 100 μL culture medium. The antibodies werediluted into the culture medium using 6-fold dilution series and 50 μLof the diluted material was added per well. Then IL3 was added to thecells at the final concentration 1 ng/mL. The final antibodyconcentration typically ranged from 6×10⁻⁸ M to 8×10⁻¹²M. Cells wereincubated at 37° C. in a humidified 5% CO₂ incubator for 3 days.Relative cell number in each well was determined by WST-8 assay asdescribed above. The relative cell number value was plotted against theantibody concentration and presented in FIG. 3. It is apparent, thatmuCD123-3, muCD123-6, muCD123-14, and 7G3 inhibit IL-3 dependentproliferation of TF-1 cells substantially and in a dose-dependentmanner, while a control non-functional anti-CD123 antibody had no sucheffect. For example, treatment with 7G3 reduced the relative cell numberto 18% at the highest antibody concentration tested, with the IC₅₀ valueof 0.33 nM. Treatment with muCD123-3 reduced the relative cell number to2% at the highest antibody concentration tested, with the IC₅₀ value of0.26 nM. Likewise, treatment with muCD123-14 or muCD123-6 reduced therelative cell number to less than 1% at the highest antibodyconcentration tested, with the IC₅₀ values of 0.08 nM or 0.05 nM,respectively. Therefore, muCD123-3, muCD123-6, and muCD123-14 inhibitIL-3 dependent proliferation of TF-1 cells to a significantly higherdegree than the 7G3 antibody.

Example 5 Binding Affinity of Murine Anti-CD123 Antibodies

Binding affinity was tested by flow cytometry using purified antibodies.FACS histograms demonstrating the binding of muCD123-3, muCD123-6,muCD123-14, and 7G3 to CD123-expressing TF-1 and HNT-34 cells are shownin FIGS. 4A and 4B, respectively. TF-1 cells (5×10⁴ cells per sample)were incubated with varying concentrations of murine antibodies in 200μL FACS buffer (DMEM medium supplemented with 2% normal goat serum). Thecells were then pelleted, washed twice, and incubated for 1 hr with 100μL of phycoerythrin (PE)-conjugated goat anti-mouse IgG-antibody(Jackson Laboratory). The cells were pelleted again, washed with FACSbuffer and resuspended in 200 μL of PBS containing 1% formaldehyde.Samples were acquired using a FACSCalibur flow cytometer with the HTSmultiwell sampler, or a FACS array flow cytometer, and analyzed usingCellQuest Pro (all from BD Biosciences, San Diego, US). For each samplethe geomean fluorescence intensity for FL2 was calculated and plottedagainst the antibody concentration in a semi-log plot. A dose-responsecurve was generated by non-linear regression and the EC₅₀ value of eachcurve, which corresponds to the apparent dissociation constant (K_(d))of each antibody, was calculated using GraphPad Prism v4 (GraphPadsoftware, San Diego, Calif.). A strong binding was observed for allantibodies tested and the K_(d) values correspond to 0.3 nM, 0.1 nM, 0.3nM, and 0.9 nM for muCD123-3, muCD123-6, muCD123-14, and 7G3 antibodies,respectively (FIG. 4A). Thus in this experiment, the binding by thesubject murine CD123 antibodies are at least 3-9 times better than thatby the 7G3 antibody.

Likewise, strong binding was also observed when another CD123-positiveacute myeloid leukemia cell line, HNT-34, was used for the same flowcytometry assay described above. The K_(d) values, calculated asdescribed above, were 0.2 nM, 0.07 nM, 0.5 nM, and 2 nM for muCD123-3,muCD123-6, muCD123-14, and 7G3, respectively (FIG. 4B). Thus in thisexperiment, the binding by the subject murine CD123 antibodies are atleast 4-28 times better than that by the 7G3 antibody.

These data demonstrate that muCD123-3, muCD123-6, and muCD123-14 havelower K_(d) (which represent higher affinity) than the 7G3 antibody toCD123-positive AML cells.

Example 6 Binding Affinity of Chimeric Anti-CD123 Antibodies

The chimeric antibodies chCD123-3, chCD123-6, and chCD123-14 wereassayed for their binding affinity to HNT-34 cells in comparison to achimeric isotype control IgG (chKTI). Flow cytometry binding assays werecarried out and analyzed as described in Example 5 using secondaryPE-conjugated goat-anti-human antibodies. FIG. 5A depicts thedose-response curves for each antibody. The value for the apparentdissociation constant (K_(d)) of each antibody was calculated usingGraphPad Prism v4 (GraphPad software, San Diego, Calif.). The data showthat chimerization only moderately affected the binding affinities ofthese antibodies. The K_(d) values for chCD123-3, chCD123-6, andchCD123-14 were 0.1 nM, 0.04 nM, and 0.2 nM, respectively. These valueswere at most 2.5 fold different from those for their murine counterpartsreported in the Example 5. As expected, the chKTI antibody did not bindto the cells at the tested concentrations (FIG. 5A).

The high affinity of chCD123-3, chCD123-6, and chCD123-14 to AML cellswas confirmed using another CD123-positive acute myeloid leukemia cellline, MOLM-13. Flow cytometry binding assays were carried out andanalyzed as described above. FIG. 5B depicts the dose-response curvesfor each antibody. The values for the apparent dissociation constant(K_(d)) of chCD123-3, chCD123-6, and chCD123-14 were 0.2 nM, 0.08 nM,and 0.2 nM, respectively. Only marginal binding was observed forchimeric isotype control IgG (chKTI antibody) at the highestconcentration tested (10 nM).

Thus, chCD123-3, chCD123-6, and chCD123-14 retain the high affinity oftheir murine counterparts.

Functional Activity of Chimeric Antibodies

chCD123-3, chCD123-6, and chCD123-14 and a non-functional anti-CD123antibody, used as a negative control, were assayed for their ability toinhibit IL3-dependent proliferation of TF-1 cells. The assays werecarried out and analyzed as described in Example 4. The treatment withchCD123-3, chCD123-6, and chCD123-14 reduced the relative cell number ina dose-dependent manner with the IC₅₀ values of 0.1 nM, 0.03, and 0.05nM, respectively (FIG. 6). The control non-functional antibody did notaffect the cell growth. Therefore, chCD123-3, chCD123-6, and chCD123-14retained the functional activity of their murine counterparts.

Example 7 Binding Affinity of Humanized Anti-CD123 Antibodies

The binding affinity of the exemplary humanized anti-CD123 antibodies,huCD123-6Gv4.7S2 and huCD123-6Gv4.7S3, to HNT-34 cells was compared tothat of their murine and chimeric counterparts, muCD123-6 and chCD123-6,respectively. The 7G3 antibody and a chimeric isotype control IgG(chKTI) were tested in parallel. Flow cytometry binding assays werecarried out and analyzed as described in Example 5. FIG. 7A depicts thedose-response curves for each antibody. These data show thathumanization did not affect the binding affinity of the antibody as theK_(d) for huCD123-6Gv4.7S2, huCD123-6Gv4.7S3, chCD123-6, and muCD123-6are approximately 0.06 nM. The apparent K_(d) for the 7G3 antibody wassignificantly higher, approximately 2 nM. The chKTI antibody did notbind to the cells at the tested concentrations. Therefore, bothhuCD123-6Gv4.7 clones retain the high affinity of the murine andchimeric counterparts and have a higher affinity (e.g., at least about30-fold higher) than the 7G3 antibody to CD123-expressing cells.

Similarly, the binding affinities of the ADC conjugates of the exemplaryhumanized huCD123-6Gv4.7 antibody were assayed using HNT-34 cells, incomparison to that of the unconjugated huCD123-6Gv4.7. Flow cytometrybinding assays were carried out and analyzed as described in Example 5using secondary PE-conjugated goat-anti-human antibodies. FIGS. 7B and7C depict the dose-response curves for each antibody and thecorresponding conjugates. The data show that conjugation only moderatelyaffected the binding affinities of these antibodies.

Functional Activity of Humanized Anti-CD123 Antibodies

The functional activity (the ability to inhibit IL3-dependentproliferation of TF-1 cells) of the exemplary humanized anti-CD123antibodies, huCD123-6Gv4.7S2 and huCD123-6Gv4.7S3, was compared to thatof their chimeric counterpart, the chCD123-6 antibody. The 7G3 antibodywas tested in parallel. The assays were carried out and analyzed asdescribed in Example 4.

The treatment with huCD123-6Gv4.7S2, huCD123-6Gv4.7S3, and chCD123-6inhibited IL-3 dependent proliferation similarly: the proliferation wascompletely inhibited at 1 nM with an IC₅₀ of 0.02 nM (FIG. 8A). However,the treatment with 7G3 did not have such a profound effect on theproliferation of the cells: the antibody IC₅₀ was 0.2 nM and it was notable to inhibit the cell proliferation completely, but only reduced thecell number to 18% at the highest concentration (10 nM).

The inhibition of TF-1 cell proliferation by huCD123-6Gv4.7S2,huCD123-6Gv4.7S3, chCD123-6, and 7G3 was IL-3 dependent, as theseantibodies had no inhibitory effect when the cells were grown in thepresence of another growth factor GM-CSF (FIG. 8B).

Therefore, huCD123-6Gv4.7 retains the functional activity of its murineand chimeric counterparts, and is significantly more active than the 7G3antibody in inhibiting IL-3 dependent proliferation.

Example 8 Epitope Mapping

The CD123 antigen, IL-3 receptor chain a (IL-3Rα), is composed of 378amino acids, containing a 306-amino acid extracellular domain involvedin IL-3 binding, a 20-amino acid transmembrane domain, and a shortcytoplasmic tail of 52 amino acids. The extracellular domain can befurther divided into an N-terminal domain (NTD) comprising a region fromthe threonine at position 19 of the mature protein (e.g., post signalpeptide cleavage), to the serine at position 100, and the cytokinerecognition motif (CRM) composed of two discrete folding domains: domain2 (amino acids 101-204) and domain 3 (amino acids 205-306). Epitopes forthe certain anti-CD123 antibodies described herein were mapped byengineering chimeric proteins utilizing combinations of theextracellular domain of IL-3Rα and the granulocyte-macrophagecolony-stimulating factor receptor α chain (GMRα), which conserves theIL-3Rα topology and shares a common β-subunit that is essential forsignaling.

IL-3Rα Variants Cloning and Expression

The extracellular domain of CD123/IL-3Rα (residues 1-306) was expressedas a histidine tagged protein. The protein sequence was codon optimizedand synthesized by Life Technologies and cloned in-frame with a10-Histidine tag in the pABLT mammalian expression vector utilizingEcoRI and HindIII restrictions sites. The extracellular domain of thecontrol GMRα protein (residue 1-325), also comprising an N-terminaldomain from the serine at position 25 to the serine at position 114 ofthe mature protein, and an CRM domain comprising the glycine at position115 to the valine at position 325, was similarly synthesized and cloned.Then the IL3Rα/GMRα chimeric receptor protein expression vectors wereconstructed by restriction digests replacing either the N-terminaldomain (residues 1-100) or the CRM domain (residues 101-306) of theIL-3Rα molecule with the corresponding sequences of the GMRα molecule.IL3Rα (1-100) encodes IL3Rα residues 1-100 fused to GMRα residues115-325, and IL3Rα (101-306) encodes GMRα residues 1-114 fused to IL3Rαresidues 101-306, as illustrated in FIG. 9A.

IL3-Rα, GMRα, and the two chimeric IL-3Rα His-tagged proteins, IL3-Rα(1-100) and IL3-Rα (101-306), were expressed via transient transfectionof HEK 293T cells, and purified from the supernatant of the transfectedcells using Ni Sepharose excel chromatography (GE healthcare) followingmanufacturer's instruction.

Antibody Binding to Various IL3Rα Constructs

The chimeric and humanized anti-IL3Rα antibodies were tested inenzyme-linked immunosorbent assay (ELISA) format for binding to theIL-3Rα proteins described above. Briefly, each His-tagged IL-3Rα proteinpurified by Ni Sepharose excel chromatography was diluted to 1 ng/mL in50 mM sodium bicarbonate buffer pH 9.6, and 100 μL was added to eachwell. After a 16 hr incubation at 4° C., the plates were washed withTris-buffered saline with 0.1% Tween-20 (TBST), then blocked with 200 μLblocking buffer (TBS with 1% BSA). Next, 100 μL of primary antibody,serially diluted in blocking buffer, was added in duplicate to the ELISAwells and incubated at room temperature for 1 hr. The plates were thenwashed 3 times with TBST before adding 100 μL of anti-human IgG(H+L)-HRP (Jackson ImmunoResearch) to each well. Once again the plateswere incubated for 1 hr at room temperature followed by three washeswith TBST. Finally, 100 μL of TMB one component HRP microwell substrate(Surmodics) was added to each well and incubated for 5 min. The reactionwas stopped by adding 100 μL stopping solution (Surmodics) andabsorbances were read at 450 nm.

Binding of the CD123 antibodies to the chimeric IL-3Rα proteins wasevaluated in comparison to the wild type IL-3Rα. FIG. 9B demonstratesthat the huCD123-6 antibody binds to both IL-3Rα and IL-3Rα (101-306)with similar sub-nanomolar affinities. Conversely, the huCD123-6antibody does not bind the GMRα construct and binding is all buteliminated for the chimeric protein IL-3Rα (1-100) construct. Theseresults indicate that huCD123-6 antibody binds primarily to the CRMdomain, with perhaps only minimal contribution from the N-terminaldomain of IL-3Rα. Similarly, the chCD123-3 antibody binds primarily tothe IL-3Rα CRM domain, albeit with a reduced binding affinity comparedto the wild type IL-3Rα (FIG. 9C). The reduction in binding affinity tochimeric receptor protein IL-3Rα (101-306) suggests a possibleinvolvement of the N-terminal domain of the IL-3Rα in CD123-3 antibodybinding. The chCD123-14 antibody however, does not recognize thechimeric receptor IL-3Rα(101-306) (FIG. 9D); rather, it binds to bothIL-3Rα and IL-3Rα (1-101) with equivalent affinity. These resultsdemonstrate that the chCD123-14 antibody binds exclusively to N-terminaldomain of IL-3Rα. In comparison, the 7G3 antibody along with the twoother commercially available antibodies, 6H6 and 9F5, also bind to theIL-3Rα N-terminal domain and wild-type IL-3Rα constructs, but do notrecognize the CRM domain of IL3Rα (FIGS. 9E, 9F, and 9G, respectively).In summary, these data demonstrate that the epitopes of the CD123-6 andCD123-3 antibodies are located primarily within the CRM domain of theIL-3Rα, and are distinct from the 7G3 antibody epitope, which isrestricted to the N-terminal domain of IL-3Rα. The CD123-14 antibodyalso binds an epitope confined within the N-terminal domain of IL-3Rα.

Example 9 Preparation of Lysine-Linked DM1 and IGN Conjugates of thehuCD123-6 Antibody a. Preparation of huCD123Gv4.7S3-sulfo-SPDB-D1

A reaction containing 2.0 mg/mL CD123-6G4.7S3 antibody and 3.5 molarequivalents of sulfo-SPDB-D1 in situ mixture by linker in 15 mM HEPES(4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid) pH 8.5 buffer and15% v/v DMA (N,N Dimethylacetamide) cosolvent was allowed to conjugatefor 3-4 hours at 25° C. The in situ mixture was prepared by reacting 3.0mM sulfo-SPDB linker with 3.9 mM of sulfonated compound D1 in DMA for 5hours at 25° C. in the presence of 20 mM N,N Diisopropylethyl amine(DIPEA).

Post-reaction, the conjugate was purified and buffer exchanged into 20mM histidine, 50 mM sodium chloride, 8.5% w/v sucrose, 0.01% Tween-20,50 μM sodium bisulfite pH 6.2 formulation buffer using NAP desaltingcolumns (Illustra Sephadex G-25 DNA Grade, GE Healthcare). Dialysis wasperformed in the same buffer for 4 hours at room temperature and thenovernight at 4° C. utilizing Slide-a-Lyzer dialysis cassettes(ThermoScientific 10,000 MWCO).

The purified conjugate was found to have a final protein concentrationof 1.2 mg/ml and an average of 2.9 molecules of compound D1 linked perantibody (by UV Vis using molar extinction coefficientsε_(330 nm)=15,484 cm⁻¹M⁻¹ and ε_(280 nm)=30, 115 cm⁻¹M⁻¹ for compoundD1, and ε_(280 nm)=207,360 cm⁻¹M⁻¹ for huCD123-6G4.7S3 antibody); 94.3%monomer (by size exclusion chromatography); and <2% unconjugatedcompound D1 (UPLC Dual column, reverse-phase HPLC analysis).

b. Preparation of huCD123-6Gv4.7S3-D2

A reaction containing 2.0 mg/mL huCD123-6Gv4.7S3 antibody and 5.0 molarequivalents of compound D2 (pretreated with 5-fold excess of sodiumbisulfite in 90:10 DMA:50 mM succinate pH 5.5 for 4 hours at 25° C.) in15 mM HEPES (4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid) pH 8.5buffer and 10% v/v DMA (N,N-Dimethylacetamide) cosolvent was incubatedfor 4 hours at 25° C.

Post-reaction, the conjugate was purified and buffer exchanged into 20mM histidine, 50 mM sodium chloride, 8.5% w/v sucrose, 0.01% Tween-20,50 μM sodium bisulfite pH 6.2 formulation buffer using NAP desaltingcolumns (Illustra Sephadex G-25 DNA Grade, GE Healthcare). Dialysis wasperformed in the same buffer for 4 hours at room temperature and thenovernight at 4° C. utilizing Slide-a-Lyzer dialysis cassettes(ThermoScientific 10,000 MWCO).

The purified conjugate was found to have a final protein concentrationof 1.2 mg/ml and an average of 2.9 molecules of compound D2 linked perantibody (by UV Vis using molar extinction coefficientsε_(330 nm)=15,484 cm⁻¹M⁻¹ and ε_(280 nm)=30, 115 cm⁻¹M⁻¹ for compoundD2, and ε_(280 nm)=207,360 cm⁻¹M⁻¹ for huCD123-6Gv4.7S3 antibody); 99.3%monomer (by size exclusion chromatography); and <2% unconjugatedcompound D2 (UPLC Dual column, reverse-phase HPLC analysis).

c. Preparation of huCD123-6Gv1.1-sulfo-SPDB-DGN462

NHS-sulfo-SPDB-sDGN462 was formed in situ by incubating 1.5 mMsulfo-SPDB linker, 1.95 mM sulfonated DGN462 (sDGN462) in DMA containing10 mM DIPEA (N,N-diisopropylethylamine) for 20 min before adding 0.9 mMMPA (3-Maleimidopropionic Acid) to quench unreacted IGN thiol for 15 minat 25° C. A reaction containing 2.5 mg/mL huCD123-6Gv1.1 antibody and7.5 molar equivalents of the resulting NHS-sulfo-SPDB-DGN462 in 15 mMHEPES (4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid) pH 8.5buffer and 15% v/v DMA cosolvent was incubated overnight at 25° C.

Post-reaction, the conjugate was purified into 20 mM Histidine, 50 mMNaCl, 8.5% sucrose, 0.01% Tween-20, 50 μM sodium bisulfite pH 6.2formulation buffer using NAP desalting columns (Illustra Sephadex G-25DNA Grade, GE Healthcare). Dialysis was performed in the same bufferover night at 4° C. utilizing Slide-a-Lyzer dialysis cassettes(ThermoScientific 10,000 MWCO).

The purified conjugate was found to have a final antibody concentrationof 0.95 mg/mL and an average of 2.8 DGN462 molecules linked per antibody(by UV-Vis using molar extinction coefficients ε_(330 nm)=15,484 cm⁻¹M⁻¹and ε_(280 nm)=30, 115 cm⁻¹M⁻¹ for DGN462, and ε_(280 nm)=207,360cm⁻¹M⁻¹ for huCD123-6Gv1.1 antibody); 99.5% monomer (by size exclusionchromatography); and 0.6% unconjugated DGN462 (by acetone precipitation,reverse-phase HPLC analysis).

d. Preparation of huCD123-6Gv1.1-D3

A reaction containing 2.0 mg/mL huCD123-6Gv1.1 antibody and 3.5 molarequivalents D3 (pretreated with 5-fold excess of sodium bisulfite in90:10 DMA:50 mM succinate pH 5.5 for 4 hours at 25° C.) in 15 mM HEPES(4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid) pH 8.5 buffer and10% v/v DMA (N,N-Dimethylacetamide) cosolvent was incubated for 4 hrs at25° C. Post-reaction, the conjugate was purified and buffer exchangedinto 20 mM histidine, 50 mM sodium chloride, 8.5% w/v sucrose, 0.01%Tween-20, 50 μM sodium bisulfite pH 6.2 formulation buffer using NAPdesalting columns (Illustra Sephadex G-25 DNA Grade, GE Healthcare).Dialysis was performed in the same buffer for 4 hrs at room temperatureand then overnight at 4° C. utilizing Slide-a-Lyzer dialysis cassettes(ThermoScientific 10,000 MWCO).

The purified conjugate was found to have a final protein concentrationof 0.9 mg/mL and an average of 3.4 D3 molecules linked per antibody (byUV-Vis using molar extinction coefficients ε_(330 nm)=15,484 cm⁻¹M⁻¹ andε_(280 nm)=30, 115 cm⁻¹M⁻¹ for D3, and ε_(280 nm)=207,360 cm⁻¹M⁻¹ forhuCD123-6Gv1.1 antibody); 96% monomer (by size exclusionchromatography); and <1% unconjugated D3 (dual column, reverse-phaseHPLC analysis).

e. Preparation of huCD123-6Rv1.1-CX1-1-maytansinoid Conjugates

Humanized anti-CD123 antibody (resurfaced huCD123-6Rv1.1) was conjugatedat lysine residues with maytansinoid payload via triglycyl, CX1-1linker. A mixture containing 5 mM triglycyl, CX1-1 heterobifunctionallinker (bearing N-hydroxysuccinimide and maleimide groups) and 6.5 mMDM1 maytansinoid in N,N-dimethylacetamide (DMA) containing 20 mMN,N-diisopropylethylamine (DIPEA) was incubated for about 20 min at roomtemperature. Unreacted DM1 was quenched with 2 mM maleimidopropionicacid (MPA) for about 20 min at room temperature before adding thereaction mixture to humanized anti-CD123 antibody (huCD123-6Rv1.1) at 4mg/mL in 140 mM EPPS buffer, pH 8, containing about 8% DMA, at an excessof about 7.6× or 15×CX1-1-DM1 adduct over antibody. The conjugationreaction mixtures were incubated overnight at 25° C., after which theconjugates were purified and buffer exchanged into 10 mM succinatebuffer, pH 5.5, containing 250 mM glycine, 0.5% sucrose, 0.01% Tween 20using NAP desalting columns (Illustra, Sephadex G-25, GE Healthcare).Dialysis was performed in the above-described succinate buffer overnightat 4° C. utilizing Slide-a-Lyzer dialysis cassettes (Thermo Scientific;10,000 molecular weight cut-off membrane).

The purified conjugates were found to contain an average of 4.3 and 6.7maytansinoid molecules linked per antibody (by UV/Vis spectrometry andsize-exclusion HPLC, using molar extinction coefficients ofε_(252 nm)=26350 cm⁻¹M⁻¹ and ε_(280 nm)=5456 cm⁻¹M⁻¹ for DM1, andε_(280 nm)=207076 cm⁻¹M⁻¹ for huCD123 antibody), 98% monomer (by sizeexclusion chromatography), and a final protein concentration of 2.1mg/mL and 1.2 mg/mL, respectively. The levels of unconjugatedmaytansinoid in purified conjugates were estimated by HPLC to be low(<1%). Mass spectrometry of deglycosylated conjugates indicated linkedmaytansinoid species.

Example 10 In Vitro Cytotoxicity Assays

The ability of antibody-drug conjugates (ADC) of huCD123-6 to kill cellsthat express CD123 on their cell surface was measured using in vitrocytotoxicity assays. The cell lines were cultured in culture medium asrecommended by the cell supplier (ATCC or DSMZ). The cells, 2,000 to10,000 in 100 μL of the culture medium, were added to each well of flatbottom 96-well plates. To block Fc receptors on the cell surface, theculture medium was supplemented with 100 nM chKTI antibody (an antibodyof the same isotype). Conjugates were diluted into the culture mediumusing 3-fold dilution series and 100 μL were added per well. Todetermine the contribution of CD123-independent cytotoxicity, CD123block (e.g., 100 nM of chCD123-6 antibody) was added to some wells priorto the conjugates. Control wells containing cells and the medium butlacking the conjugates, as well as wells contained medium only, wereincluded in each assay plate. Assays were performed in triplicate foreach data point. The plates were incubated at 37° C. in a humidified 6%CO₂ incubator for 4 to 7 days. Then the relative number of viable cellsin each well was determined using the WST-8 based Cell Counting Kit-8(Dojindo Molecular Technologies, Inc., Rockville, Md.) as described inExample 4. The apparent surviving fraction of cells in each well wascalculated by first correcting for the medium background absorbance, andthen dividing each value by the average of the values in the controlwells (non-treated cells). The surviving fraction of cells was plottedagainst conjugate concentration in semi-log plots.

The results from a typical cytotoxicity assay are shown in FIG. 10. TheAML cell line OCI-AML4 can proliferate without growth factors in culturemedium. The cells were treated with the maytansinoid conjugatehuCD123-6Rv1.1-CX1-1-DM1. The treatment resulted in dose-dependent cellkilling with the IC₅₀ value of 0.07 nM. To assess whether the killingwas due to CD123 expression, the antigen was blocked by an excess ofunconjugated chCD123-6 antibody (500 nM) and potency of the conjugatewas tested on the cells. The later treatment did not affect viability ofthe cells at the concentrations lower than 3 nM and had only moderateeffect on the cell viability at 10 nM (the highest concentrationtested). Thus, the huCD123-6Rv1.1-CX1-1-DM1 conjugate demonstrates highCD123-dependent cytotoxicity on OCI-AML4 cells.

Cytotoxicity of the conjugates of the huCD123-6 antibody linked withother cytotoxic agents (DGN462, D3, D1, and D2) via lysines was alsotested in vitro. Fifteen CD123-positive cell lines of different origin(AML, B-ALL, CML and NHL) were used in the study (table immediatelybelow). The majority of the cell lines were derived from patientscarrying a malignancy with at least one negative prognostic factor(e.g., overexpression of P-glycoprotein, overexpression of EVI1, p53alterations, DNMT3A mutation, FLT3 internal tandem duplication). Theconjugates demonstrated high potency on these cell lines with IC₅₀values ranging from sub-pM to low nM (table immediately below, FIG.11A).

In vitro cytotoxicity of the various lysine-linked huCD123-6-IGNconjugates on CD123-positive cell lines of different origin IC₅₀, M Cellline Negative prognostic DGN462 D3 D2 D1 name Origin factor ADC ADC ADCADC THP1 AML p53 deletion 1.9E−10 3.0E−11 6.7E−12 5.8E−11 SHI-1 AML p53gene alterations 1.7E−10 2.9E−11 1.3E−11 3.2E−11 KO52 AML p53 mutant,Pgp 3.9E−10 2.4E−10 1.4E−11 4.1E−10 overexpression KASUMI-3 AML EVI1 andPgp 2.8E−09 2.3E−11 9.8E−12 1.4E−10 overexpression KG-1 AML p53 mutant,Pgp 8.5E−10 6.6E−09 2.2E−10 4.1E−09 overexpression OCI- AML DNMT3Amutation 1.4E−10 1.0E−10 8.8E−11 2.1E−10 AML2 HNT-34 AML MECOM (EVI1)2.3E−11 3.2E−12 2.0E−12 5.9E−12 overexpression MV4-11 AML FLT3 internaltadem 1.6E−12 5.4E−13 5.6E−13 1.3E−12 duplication MOLM-13 AML FLT3internal tadem 2.2E−12 4.6E−13 4.9E−13 1.2E−12 duplication EOL-1 AML9.0E−12 3.3E−12 2.5E−12 4.7E−12 MOLM-1 CML EVI1 and Pgp 1.1E−09 7.1E−112.9E−11 2.1E−10 overexpression KOPN8 B-ALL 5.3E−11 2.2E−11 1.1E−113.0E−11 JM-1 B-ALL 3.2E−10 1.7E−10 2.4E−11 4.1E−10 KCL-22 CML 2.0E−099.5E−10 3.0E−11 2.9E−10 Granta519 NHL 1.2E−11 2.1E−12 1.2E−12

The above data seems to suggest that the B-ALL cell lines (KOPN8 andJM-1) are very sensitive to the IGN compounds. To further validate thisfinding, cytotoxicity of the conjugates of the huCD123-6Gv4.7 antibodylinked with D1 or D2, via lysines or cysteines, was tested using thesame B-ALL cell lines, KOPN8 and JM-1, plus an additional B-ALL cellline “380 cells.” Negative controls using conjugates with an antibodythat does not bind to these B-ALL cell lines—KTI—were included in theassays. The results in FIG. 11B confirmed the above finding.

The above table also shows that the huCD123-IGN compounds are highlyactive against the various AML cell lines. Representative data usingLys- and Cys-linked IGN compounds are shown in FIG. 11C. Consistent withthe data shown in FIG. 11C, additional data (shown in the table belowand FIG. 21) on cytotoxicity of the Cys-linked conjugate(huCD123-6Gv4.7-CysMab-D5) demonstrate that the conjugate is highlypotent against various CD123-positive AML cell lines, including thosewith poor prognostic factors.

Fold-specificity IC₅₀ control huCD123- ADC/IC₅₀ Poor prognosticCysMab-D5 huCD123- Cell line factor IC₅₀ pMol CysMab-D5 CD123 negativecell lines Namalwa 10000 1 K562 8000 1 CD123 positive AML cell linesSKM-1 P53 7 57 KO52 20 100 EOL-1 2 1000 UCSD-AML1 EVI OX 1 400 KG-1 P53,MDR1 60 95 THP-1 P53 30 167 SH1-1 P53 6 >3,333 MOLM-1 MDR1 and EVI1 OX120 >167 Molm-13 FLT/ITD 0.5 2000 MV4-11 FLT/ITD 1 2000 KASUMI-3 P53 andMDR1 3 100 HNT-34 EVI1 OX 1 1100

Interestingly, preliminary data suggests that Cys-linked D5 conjugateappears to be particularly potent on AML progenitor cells, even whencompared to potent conjugates of Lys-linked D2 or Lys-linked D1. SeeFIG. 11D, in which 9 AML patient samples were used to test the potencyof the various IGN conjugates of the invention.

Additional in vitro cytotoxicity studies show that the Cys-linked D5conjugate has 74 fold higher activity than Mylotarg in unselected AMLpatient samples. See FIG. 18.

In addition, FIG. 11E and FIG. 19 show that the Cys-linked huCD123 D5conjugate kills normal blood cells at concentrations that are >100-foldhigher than those needed to kill AML progenitors. In comparison,Mylotarg (gemtuzumab ozogamicin) does not exhibit such preferentialkilling effect, with only 10 fold difference in cytotoxicity betweennormal progenitor cells and AML progenitor cells. In FIG. 19, additionalAML patient samples were tested. In addition, the huCD123 conjugate ofD5′, a DNA cross-linker, was also tested and it only exhibits 6 folddifference in cytotoxicity between normal progenitor cells and AMLprogenitor cells (see FIG. 19).

Example 11 In Vitro Potency of Lysine-Linked IGN Conjugates on PrimaryAML Patient Samples

The ability of the various lysine-linked huCD123-6-IGN conjugates tokill primary AML cells was measured using colony forming unit (CFU)assays. Frozen Peripheral Blood Mononuclear Cells (PBMC) and Bone MarrowMononuclear Cells (BMMC) from patients with AML were purchased fromConversant Biologics Inc. (Huntsville, Ala.) and AllCells, LLC (Alameda,Calif.). The cells were thawed as recommended by the suppliers, washedand resuspended in the RPMI culture medium (RPMI-1640, 10% fetal bovineserum, 50 ng/mL SCF and 50 ng/mL FLT3L). To block Fc receptors on thecell surface, the culture medium was supplemented with 100 nM chKTIantibody (an antibody of the same isotype). The cells, 200,000 in 150 μLof the culture medium, were added to each well of flat bottom 96-wellplates. Conjugates were diluted into the medium using 10-fold dilutionseries and 50 μL were added per well. Control wells contained cells andthe medium but lacked the conjugates. The plates were incubated at 37°C. in a humidified 6% CO₂ incubator for 18 hours. Then the cells weretransferred to tubes containing 2.2 mL of METHOCULT™ H4534 without EPO(StemCell Technologies, Vancouver, BC), mixed, and the mixtures weretransferred to 6-well plates. The plates were incubated at 37° C. in ahumidified 6% CO₂ incubator until colonies formed (usually 10 to 16days) and were counted. Percent inhibition of colony formation wasdetermined by comparing the counts in the conjugate-treated samples bythat in the non-treated control. The percent colony inhibition wasplotted against the conjugate concentration and the conjugateconcentration that inhibits 90% of the colony formation (IC₉₀) wasdetermined from the curves.

The result from a typical CFU assay for one primary patient sample ispresented in FIG. 12A. The huCD123-6-IGN conjugates demonstrated adose-dependent cytotoxicity with the IC₉₀ values of 0.1 nM, 0.01 nM,0.03 nM, and 0.012 nM for huCD123-6Gv1.1-sSPDB-DGN462,huCD123-6Gv1.1-D3, huCD123-6Gv1.1-sSPDB-D1 and huCD123-6Gv1.1-D2,respectively. FIG. 12B shows the IC₉₀ values for all AML patient samplestreated with the conjugates. The median IC₉₀ values for each conjugateare presented as solid lines and equal to 2 nM, 0.1 nM, 0.03 nM, and0.02 nM for huCD123-6Gv1.1-sSPDB-DGN462, huCD123-6Gv1.1-D3,huCD123-6Gv1.1-sSPDB-D1, and huCD123-6Gv1.1-D2, respectively. Thus, thelysine-linked huCD123-6-IGN conjugates demonstrated high potency onsamples from AML patients.

Example 12 Preparation of Ser Site-Specific Conjugates of the huCD123-6Antibody a) N-Terminal Antibody Conjugation—a Two-Step Approach

huCD123-6Gv4.6/7S3 antibody (having huCD123-6Gv4 LCVR (SEQ ID NO: 37,including an engineered N-terminal Ser) and HCVR Gv6/7 (SEQ ID NO: 34))([1], in Scheme 1 as shown in FIG. 15; 3 mg/mL in PBS, pH7.4) wastreated with 5 mM aqueous sodium periodate (50 equivalents, 25° C., 30minutes). The mixture was then buffer exchanged through a NAP desaltingcolumn (Illustra Sephadex G-25 DNA Grade, GE Healthcare) into sodiumacetate buffer, pH5.0.

The resulting solution was treated with 4-Aminophenethyl alcohol (100 mMin DMA [N,N-Dimethylacetamide]) to 10% v/v cosolvent. HeterobifunctionalLinker1 ([3] in Scheme 1; 5 equivalents) was subsequently introduced,and the reaction vessel was sealed and incubated at 37° C. for 24 hours.

The mixture was then buffer exchanged through a NAP desalting column(Illustra Sephadex G-25 DNA Grade, GE Healthcare) into HEPES(4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid), pH8.5 buffer. Thesolution was then adjusted with DMA (N,N-Dimethylacetamide) cosolvent(10% v/v), and treated with sulfonated DGN462 (sDGN462) ([5], Scheme 1;free thiol; 5 equivalents), at 25° C. for 6 hours.

The resulting conjugate was buffer exchanged into 250 mM Glycine, 10 mMHistidine, 1% sucrose, 0.01% Tween-20, 50 μM sodium bisulfiteformulation buffer at pH 6.2 using a NAP filtration column (IllustraSephadex G-25 DNA Grade, GE Healthcare). Dialysis was performed in thesame buffer for 4 hours at 25° C. utilizing Slide-a-Lyzer dialysiscassettes (ThermoScientific 10,000 MWCO).

The purified conjugate ([6], Scheme 1) was found to have a homogenousaverage of two DGN462 molecules linked per antibody (via Q-ToF MassSpectrometry), >98% monomer (via Size Exclusion Chromatography), <2%free drug (via acetone precipitated reverse-phase HPLC analysis), and afinal protein concentration of 0.18 mg/mL (via UV-Vis using molarextinction coefficients ε₂₈₀=213320 M⁻¹ cm⁻¹ for the huCD123-6Gv4.6/7S3antibody).

b) N-Terminal Antibody Conjugation—IGN Direct Link

The engineered N-terminal Ser-containing huCD123-6Gv4.7S2 antibody,engineered with an N-terminal serine on the heavy chain(huCD123-6Gv4.7S2, which comprises heavy chain sequence SEQ ID NO: 53 inwhich Xaa is Val, and light chain sequence SEQ ID NO: 51) ([1] in Scheme2, FIG. 16; 3 mg/mL in PBS, pH7.4) was treated with 5 mM aqueous sodiumperiodate (50 molar equivalents) at 25° C. for 30 minutes. The mixturewas then buffer exchanged through a NAP desalting column (IllustraSephadex G-25 DNA Grade, GE Healthcare) into sodium acetate buffer,pH5.0.

The resulting solution was treated with p-phenylenediamine (100 mM inDMA [N,N-Dimethylacetamide]) to 10% v/v cosolvent. Then, an in situsulfonated-D8 (or sD8) ([3], Scheme 2; 5 molar equivalents) wassubsequently introduced, and the reaction vessel was sealed andincubated at 37° C. for 24 hours.

The mixture was then buffer exchanged through a NAP desalting column(Illustra Sephadex G-25 DNA Grade, GE Healthcare) into 250 mM Glycine,10 mM Histidine, 1% sucrose buffer at pH 6.2. Dialysis was performed inthe same buffer for 4 hours at 25° C., utilizing Slide-a-Lyzer dialysiscassettes (ThermoScientific 10,000 MWCO).

The purified conjugate ([4], Scheme 2) was found to have a homogenousaverage of two D8 molecules linked per antibody (via Q-ToF MassSpectrometry), >96% monomer (via Size Exclusion Chromatography), <3%free drug (via HISEP reverse-phase HPLC analysis), and a final proteinconcentration of 1.4 mg/mL (via UV-Vis using molar extinctioncoefficients ε₂₈₀=213320 M⁻¹ cm⁻¹ for the huCD123-6Gv4.7S2 antibody).

The in situ sulfonated-D8 (or sD8) described above was preparedaccording to the following procedure: The D8 reagent, as a lyophilized,white solid, was dissolved in DMA (N,N-Dimethylacetamide) to a 10-20 mMstock concentration solution. Fresh sodium bisulfite (500 mM solution inwater, 5 molar equivalents) was added and the resulting solution reactedfor 4-6 hours at 25° C. before a 15 hour hold step at 4° C. A furtheraliquot of fresh sodium bisulfite (500 mM solution in water, 2 molarequivalents) was introduced and allowed to react for 4 hours at 25° C.before storage at −80° C. until further use.

c) N-Terminal Antibody Conjugation—Two-Step Protocol for CD123-6Gv4.7

The huCD123-6Gv4.7S3 antibody (see above) engineered with an N-terminalserine on the light chain (huCD123-6Gv4.7S3) ([1] in Scheme 3, FIG. 17;3 mg/mL in PBS, pH7.4) was treated with 5 mM aqueous sodium periodate(50 molar equivalents) at 25° C. for 30 minutes. The mixture was thenbuffer exchanged through a NAP desalting column (Illustra Sephadex G-25DNA Grade, GE Healthcare) into sodium acetate buffer, pH5.0.

The resulting solution was treated with 4-Aminophenethyl alcohol (100 mMin DMA [N,N-Dimethylacetamide]) to 10% v/v cosolvent. HeterobifunctionalLinker1 ([3], Scheme 3; 5 molar equivalents) was subsequentlyintroduced, and the reaction vessel was sealed and incubated at 37° C.for 24 hours.

The mixture was then buffer exchanged through a NAP desalting column(Illustra Sephadex G-25 DNA Grade, GE Healthcare) into HEPES(4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid) pH8.5 buffer. Thesolution was then adjusted with DMA (N,N-Dimethylacetamide) cosolvent(10% v/v), and treated with sulfonated-D1 (or sD1) ([5], Scheme 3; freethiol; 5 molar equivalents) at 25° C. for 6 hours.

The resulting conjugate was buffer exchanged into 250 mM Glycine, 10 mMHistidine, 1% sucrose, 0.01% Tween-20, 50 μM sodium bisulfiteformulation buffer at pH 6.2, using a NAP filtration column (IllustraSephadex G-25 DNA Grade, GE Healthcare). Dialysis was performed in thesame buffer for 4 hours at 25° C., utilizing Slide-a-Lyzer dialysiscassettes (ThermoScientific 10,000 MWCO).

The purified conjugate ([6], Scheme 3) was found to have an average of2.0 molecules of D1 linked per antibody (via Q-ToF MassSpectrometry), >96% monomer (via Size Exclusion Chromatography), <3%free drug (via acetone precipitated reverse-phase HPLC analysis), and afinal protein concentration of 0.4 mg/mL (via UV-Vis using molarextinction coefficients ε₂₈₀=213320 M⁻¹ cm⁻¹ for the huCD123-6Gv4.7S3antibody).

Example 13 In Vitro Cytotoxicity of Site-Specific Conjugates of thehuCD123-6 Antibody

The ability of site-specific conjugates of huCD123-6 with the variousIGN compounds (huCD123-6Gv4.6-CysMab-D5 and huCD123-6Rv1.1S2-SeriMab-D8)to kill cells that express CD123 on their cell surface was compared tothat of the lysine-linked conjugates containing the matching antibodyand the payload (huCD123-6Gv4.6-D2 and huCD123-6Rv1.1-D2) using in vitrocytotoxicity assays. The cytotoxicity assays were carried out andanalyzed as described in Example 10.

The huCD123-6Gv4.6-CysMab-D5 conjugate (in which thehuCD123-6Gv4.6-CysMab has an Ig heavy chain sequence of SEQ ID NO: 54,and a light chain sequence of SEQ ID NO: 51) was at least as active asthe lysine-linked huCD123-6Gv4.6-D2 conjugate (in which thehuCD123-6Gv4.6 antibody has an Ig heavy chain sequence of SEQ ID NO: 50,and a light chain sequence of SEQ ID NO: 51) on multiple cell lines.Several examples of the cytotoxicity assay using the AML cell lineEOL-1, the B-ALL cell line KOPN-8 and the CML cell line MOLM-1 are shownin FIGS. 13A-13C, respectively. Both conjugates killed the cells in adose-dependent manner with the IC₅₀ values of approximately 0.002 nM,0.005 nM, and 0.02 nM for EOL-1 cells, KOPN-8 cells and MOLM-1 cells,respectively. The killing was CD123-dependent as the conjugates were atleast 100 fold less toxic to the cells when the CD123 antigen wasblocked by the unconjugated chCD123-6 antibody.

The huCD123-6Rv1.1S2-SeriMab-D8 conjugate (in which the resurfacedhuCD123-6Rv1.1S2 antibody has an Ig heavy chain sequence of SEQ ID NO:60 except that the N-terminal residue is Ser, and a light chain sequenceof SEQ ID NO: 61) maintained target (CD123) binding, and was at least asactive as the lysine-linked huCD123-6Rv1.1-D2 conjugate (in which theresurfaced huCD123-6Rv1.1 antibody has an Ig heavy chain sequence of SEQID NO: 60, and a light chain sequence of SEQ ID NO: 61) on multiple celllines. Several examples of the cytotoxicity assay using the AML celllines SHI-1 and HNT-34, as well as the CML cell line MOLM-1 are shown inFIGS. 14A-14C, respectively. Both conjugates killed the cells in adose-dependent manner with the IC₅₀ values of approximately 0.01 nM,0.002 nM, and 0.03 nM for SHI-1 cells, HNT-34 cells, and MOLM-1 cells,respectively. The killing was CD123-dependent as the conjugates were atleast 100 fold less toxic to the cells when the CD123 antigen wasblocked by the unconjugated huCD123-6 antibody.

In another experiment, it was found that Ser-linked DGN462 compound withhuCD123 antibody has 3-fold higher antigen-specific potency than lysinelinked version with higher DAR (data not shown).

Example 14 In Vivo Efficacy Studies Using the CD123-IGN Conjugates inthe MV4-11 AML Subcutaneous Mice Model

Subcutaneously implanted tumor cells represent a convenient means totest novel potential anti-cancer drugs in vivo. A large variety of humanand murine cell lines derived from both solid tumors and leukemias,covering a wide range of tumor genotypes and phenotypes, have beenadapted to grow in a murine host, and thus allow testing of a subjecttherapeutic agent in the appropriate tumor model.

A subcutaneous acute myeloid leukemia model (AML), as outlined in theprotocol below, is used to test the efficacy of the subject CD123antibody drug conjugates (ADCs) for their ability to decrease tumorburden in vivo. Specifically, female SCID mice are each inoculatedsubcutaneously in the right flank with about 1×10⁷ MV4-11 cells, a humanAML cell line. On day 14 post-inoculation, mice are randomly dividedinto groups based on tumor volume, and treated with 400 mg/kg of humanIgG by intraperitoneal injection to block Fc receptors on MV4-11 cells.The subject anti-D123 ADCs or non-targeting antibody controls(chKTI-lysine linked-D1, chKTI-lysine linked D2, and huKTI-CysMab linkedD2 in which the huKTI antibody has an engineered Cys at a positioncorresponding to the 5^(th) to the last residue of SEQ ID NO: 54) areadministered intravenously once, on day 15 post-inoculation, at a doseof 1 or 3 μg/kg. Mice are treated with 100 mg/kg of human IgG again onday 20 post-inoculation. Animals are monitored daily, and tumor volumeis measured twice weekly. The treatment groups and control groups arelisted below with the respective doses.

Dose (μg/kg) (Actual dose Group Treatment (μg/kg)) Route and schedule 1Vehicle — i.v., ×1 2 huCD123- sSPDB-D1 1 (0.78) i.v., ×1 3huCD123-lysine linked-D2 1 (0.89) i.v., ×1 4 huCD123-CysMab-D5 1 (0.91)i.v., ×1 5 huCD123-sSPDB-D1 3 (2.35) i.v., ×1 6 huCD123-lysine linked-D23 (2.69) i.v., ×1 7 huCD123-CysMab-D5 3 (2.88) i.v., ×1 8 chKTI-sSPDB-D13 i.v., ×1 9 chKTI-lysine linked-D2 3 i.v., ×1 10 huKTI-CysMab-D5 1(0.91) i.v., ×1 11 huKTI-CysMab-D5 3 (2.88) i.v., ×1

The huCD123 antibody used in the study is the humanized huCD123-6Gv4.7antibody, which is linked to D1 or D2 through Lys linkage, or throughengineered Cys-linkage as described herein above. The Lys-linkedchimeric KTi antibody-based IGN conjugates and the Cys-linked human KTiantibody-based IGN conjugates are also included as controls. The latterKTi CysMab antibody has an engineered Cys in the heavy chain CH3 domainat a position corresponding to the 5^(th) to the last residue of SEQ IDNO: 54.

Preliminary data shows that the Lys- and Cys-linked IGN compounds arehighly active against the MV4-11 xenograft tumors in the in vivo mousemodel (see, FIG. 20).

Example 15 Preparation of Cys Site-Specific Conjugates of the huCD123-6Antibody huCD123-6Gv1.1S2-CysMab-D7

huCD123-6Gv1.152-CysMab is a CDR grafted humanized antibody with LCVRsequences of SEQ ID NO: 33 and HC sequence of SEQ ID NO: 48 (except thatthe first residue is Ser), and including an engineered Cys correspondingto the 5^(th) to the last residue of SEQ ID NO: 54 or 56.

This huCD123 antibody bearing two unpaired cysteine residues in thereduced state was prepared using standard procedures. To a solution ofthis intermediate in phosphate buffered saline (PBS), 5 mMN,N,N′,N′-ethylenediaminetetracetic acid (EDTA) pH 6.0 was addedN,N-dimethylacetamide (DMA), propylene glycol, and 10 molar equivalentsof D7 as a stock solution in DMA to give a reaction mixture with a finalsolvent composition of 2% v/v DMA and 38% v/v propylene glycol in PBS 5mM EDTA pH 6.0. The reaction was allowed to proceed for 24 hours at 25°C.

The conjugate was purified into 20 mM histidine, 50 mM sodium chloride,8.5% sucrose, 0.01% Tween-20, 50 μM sodium bisulfite pH 6.2 formulationbuffer using Sephadex G25 desalting columns, concentrated byultrafiltration through a membrane with 10 kDa molecular weight cutoff,and filtered through a 0.22 μm syringe filter. The conjugate was thendialyzed against the same buffer using a membrane with 10 kDa molecularweight cutoff.

The conjugate was found to have 2 mol D7/mol antibody by UV-Vis; 97.2%monomer by SEC; and 1.9% unconjugated D7 by SEC/reverse-phase HPLC.LC-MS of the deglycosylated conjugate is not shown.

huCD123-6Gv4.7-CysMab-D5

huCD123-6Gv4.7-CysMab is a CDR grafted humanized antibody with LCVRsequences of SEQ ID NO: 34 (in which Xaa is Val) and HCVR sequence ofSEQ ID NO: 35, and including an engineered Cys corresponding to the5^(th) to the last residue of SEQ ID NO: 54 or 56.

This huCD123 antibody bearing two unpaired cysteine residues in thereduced state was prepared using standard procedures. To a solution ofthis intermediate in phosphate buffered saline (PBS), 5 mMN,N,N′,N′-ethylenediaminetetracetic acid (EDTA) pH 6.0 was addedN,N-dimethylacetamide (DMA), propylene glycol, and 10 molar equivalentsof D5 as a stock solution in DMA to give a reaction mixture with a finalsolvent composition of 2% v/v DMA and 38% v/v propylene glycol in PBS 5mM EDTA pH 6.0. The reaction was allowed to proceed for 24 hours at 25°C.

The conjugate was purified into 20 mM histidine, 50 mM sodium chloride,8.5% sucrose, 0.01% Tween-20, 50 μM sodium bisulfite pH 6.2 formulationbuffer using Sephadex G25 desalting columns, concentrated byultrafiltration through a membrane with 10 kDa molecular weight cutoff,and filtered through a 0.22 μm syringe filter. The conjugate was thendialyzed against the same buffer using a membrane with 10 kDa molecularweight cutoff.

The conjugate was found to have 2 mol D5/mol antibody by UV-Vis and94.8% monomer by SEC. LC-MS of the deglycosylated conjugate is notshown.

huCD123-6Gv4.7-CysMab-D4

The above huCD123 antibody bearing two unpaired cysteine residues in thereduced state was prepared using standard procedures. To a solution ofthis intermediate in phosphate buffered saline (PBS), 5 mMN,N,N′,N′-ethylenediaminetetracetic acid (EDTA) pH 6.0 was addedN,N-dimethylacetamide (DMA), propylene glycol, and 5 molar equivalentsof D4 as a stock solution in DMA to give a reaction mixture with a finalsolvent composition of 2% v/v DMA and 38% v/v propylene glycol in PBS 5mM EDTA pH 6.0. The reaction was allowed to proceed for 6 hours at 25°C.

The conjugate was purified into 20 mM histidine, 50 mM sodium chloride,8.5% sucrose, 0.01% Tween-20, 50 μM sodium bisulfite pH 6.2 formulationbuffer using Sephadex G25 desalting columns, concentrated byultrafiltration through a membrane with 10 kDa molecular weight cutoff,and filtered through a 0.22 μm syringe filter. The conjugate was thendialyzed against the same buffer using a membrane with 10 kDa molecularweight cutoff.

The conjugate was found to have 1.8 mol D4/mol antibody by UV-Vis and97.4% monomer by SEC. LC-MS of the deglycosylated conjugate is notshown.

Example 16 Synthesis of Compound D1

Compound 1a:

To a stirred solution of (5-amino-1,3-phenylene)dimethanol (1.01 g, 6.59mmol) in anhydrous dimethylformamide (16.48 mL) and anhydroustetrahydrofuran (16.48 ml) was added4-methyl-4-(methyldisulfanyl)pentanoic acid (1.281 g, 6.59 mmol),N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (2.53 g,13.19 mmol), and 4-dimethylaminopyridine (0.081 g, 0.659 mmol). Theresulting mixture was stirred for 18 hours at room temperature. Thereaction was quenched with saturated ammonium chloride solution andextracted with ethyl acetate (3×50 mL). The organic extracts were washedwith water and brine, then dried over anhydrous sodium sulfate. Thesolution was filtered and concentrated in vacuo and the resultingresidue was purified by silica gel chromatography (Ethylacetate/Hexanes) to obtain compound 1a as a white solid (0.70 g, 32%yield). ¹H NMR (400 MHz, DMSO-d6: δ 9.90 (s, 1H), 7.43 (s, 2H), 6.93 (s,1H), 5.16 (t, 2H, J=5.7 Hz), 4.44 (d, 4H, J=5.7 Hz), 2.43 (s, 3H),2.41-2.38 (m, 2H), 1.92-1.88 (m, 2H), 1.29 (s, 6H). MS (m/z), found330.0 (M+1)⁺.

Compound 1b:

To a cooled (−10° C.) solution of compound 1a (219 mg, 0.665 mmol) inanhydrous dichloromethane (6.65 mL) was added triethylamine (463 μl,3.32 mmol) followed by dropwise addition of methanesulfonic anhydride(298 mg, 1.662 mmol). The mixture stirred at −10° C. for 2 hours, thenthe mixture was quenched with ice water and extracted with cold ethylacetate (2×30 mL). The organic extracts were washed with ice water,dried with anhydrous sodium sulfate, filtered and concentrated underreduced pressure to obtain the crude dimesylate.

The crude dimesylate (227 mg, 0.467 mmol) and IGN (orindolinobenzodiazepine) monomer A (303 mg, 1.028 mmol) were dissolved inanhydrous DMF (3.11 mL). Potassium carbonate (161 mg, 1.169 mmol) wasadded and the mixture stirred for 18 hours at room temperature.Deionized water was added and the resulting precipitate was filtered andrinsed with water. The solid was re-dissolved in dichloromethane andwashed with water. The organic layer was dried with anhydrous magnesiumsulfate, filtered, and concentrated. The crude residue was purified bysilica gel chromatography (Methanol/Dichloromethane) to give compound 1b(227 mg, 36% yield). MS (m/z), found 882.5 (M+1)⁺.

Compound 1c:

To a suspension of compound 1b (227 mg, 0.167 mmol) in anhydrous1,2-dichloroethane (3.346 mL) was added sodium triacetoxyborohydride(37.3 mg, 0.167 mmol). The mixture was stirred at room temp for one hourupon which it was quenched with saturated ammonium chloride solution.The mixture was extracted with dichloromethane and washed with brine.The organic layer was dried with anhydrous magnesium sulfate, filteredand concentrated. The crude residue was purified by RP-HPLC (C18,Water/Acetonitrile). Fractions containing desired product were extractedwith dichloromethane, dried with anhydrous magnesium sulfate, filteredand concentrated to give compound 1c (35 mg, 19% yield). MS (m/z), found884.3 (M+1)⁺.

Compound 1d:

To a solution of compound 1c (18 mg, 0.017 mmol) in acetonitrile (921μL) and methanol (658 μL) was added tris(2-carboxyethyl)phosphinehydrochloride (17.51 mg, 0.060 mmol) (neutralized with saturated sodiumbicarbonate solution (0.2 mL) in sodium phosphate buffer (132 μL, 0.75M, pH 6.5). The mixture was stirred at room temperature for 3.5 hours,then diluted with dichloromethane and deionized water. The organic layerwas separated, washed with brine, dried with anhydrous sodium sulfate,filtered and concentrated under reduced pressure to obtain the crudethiol. MS (m/z), found 838.3 (M+1)⁺.

The crude thiol (15.5 mg, 0.018 mmol) was dissolved in 2-propanol (1.23mL). Deionized water (617 μL) and sodium bisulfite (5.77 mg, 0.055 mmol)were added and the mixture stirred for five hours at room temperature.The reaction was frozen in an acetone/dry ice bath, lyophilized, andpurified by RP-HPLC (C18, deionized water/acetonitrile). Fractionscontaining desired product were frozen and lyophilized to give compound(12S,12aS)-9-((3-(4-mercapto-4-methylpentanamido)-5-((((R)-8-methoxy-6-oxo-11,12,12a,13-tetrahydro-6H-benzo[5,6][1,4]diazepino[1,2-a]indol-9-yl)oxy)methyl)benzyl)oxy)-8-methoxy-6-oxo-11,12,12a,13-tetrahydro-6H-benzo[5,6][1,4]diazepino[1,2-a]indole-12-sulfonicacid (compound sulfonated-D1 (sD1)) (6.6 mg, 39% yield). MS (m/z), found918.2 (M−1)⁻.

Example 17 Synthesis of 2,5-dioxopyrrolidin-1-yl6-(((S)-1-(((S)-1-((3-((((S)-8-methoxy-6-oxo-11,12,12a,13-tetrahydro-6H-benzo[5,6][1,4]diazepino[1,2-a]indol-9-yl)oxy)methyl)-5-((((R)-8-methoxy-6-oxo-12a,13-dihydro-6H-benzo[5,6][1,4]diazepino[1,2-a]indol-9-yl)oxy)methyl)phenyl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)amino)-6-oxohexanoate,compound D2

Step 1:

(S)-2-(((benzyloxy)carbonyl)amino)propanoic acid (5 g, 22.40 mmol) and(S)-tert-butyl 2-aminopropanoate hydrochloride (4.48 g, 24.64 mmol) weredissolved in anhydrous DMF (44.8 mL). EDC.HCl (4.72 g, 24.64 mmol), HOBt(3.43 g, 22.40 mmol), and DIPEA (9.75 mL, 56.0 mmol) were added. Thereaction stirred under argon, at room temperature, overnight. Thereaction mixture was diluted with dichloromethane and then washed withsaturated ammonium chloride, saturated sodium bicarbonate, water, andbrine. The organic layer was dried over sodium sulfate and concentrated.The crude oil was purified via silica gel chromatography (Hexanes/EthylAcetate) to yield compound 2a (6.7 g, 85% yield). ¹H NMR (400 MHz,CDCl₃): δ 7.38-7.31 (m, 5H), 6.53-6.42 (m, 1H), 5.42-5.33 (m, 1H), 5.14(s, 2H), 4.48-4.41 (m, 1H), 4.32-4.20 (m, 1H), 1.49 (s, 9H), 1.42 (d,3H, J=6.8 Hz), 1.38 (d, 3H, J=7.2 Hz).

Step 2:

Compound 2a (6.7 g, 19.12 mmol) was dissolved in methanol (60.7 mL) andwater (3.03 mL). The solution was purged with argon for five minutes.Palladium on carbon (wet, 10%) (1.017 g, 0.956 mmol) was added slowly.The reaction was stirred overnight under an atmosphere of hydrogen. Thesolution was filtered through Celite, rinsed with methanol andconcentrated. It was azeotroped with methanol and acetonitrile and theresulting oil was placed directly on the high vacuum to give compound 2b(4.02 g, 97% yield) which was used directly in the next step. ¹H NMR(400 MHz, CDCl₃): δ 7.78-7.63 (m, 1H), 4.49-4.42 (m, 1H), 3.55-3.50 (m,1H), 1.73 (s, 2H), 1.48 (s, 9H), 1.39 (d, 3H, J=7.2 Hz), 1.36 (d, 3H,J=6.8 Hz).

Step 3:

Compound 2b (4.02 g, 18.59 mmol) and mono methyladipate (3.03 mL, 20.45mmol) were dissolved in anhydrous DMF (62.0 mL). EDC.HCl (3.92 g, 20.45mmol), HOBt (2.85 g, 18.59 mmol) and DIPEA (6.49 mL, 37.2 mmol) wereadded. The mixture was stirred overnight at room temperature. Thereaction was diluted with dichloromethane/methanol (150 mL, 5:1) andwashed with saturated ammonium chloride, saturated sodium bicarbonate,and brine. It was dried over sodium sulfate, filtered and stripped. Thecompound was azeotroped with acetonitrile (5×), then pumped on the highvacuum at 35° C. to give compound 2c (6.66 g, 100% yield). The crudematerial was taken onto next step without purification. ¹H NMR (400 MHz,CDCl₃): δ 6.75 (d, 1H, J=6.8 Hz), 6.44 (d, 1H, J=6.8 Hz), 4.52-4.44 (m,1H), 4.43-4.36 (m, 1H), 3.65 (s, 3H), 2.35-2.29 (m, 2H), 2.25-2.18 (m,2H), 1.71-1.60 (m, 4H), 1.45 (s, 9H), 1.36 (t, 6H, J=6.0 Hz).

Step 4:

Compound 2c (5.91 g, 16.5 mmol) was stirred in TFA (28.6 mL, 372 mmol)and deionized water (1.5 mL) at room temperature for three hours. Thereaction mixture was concentrated with acetonitrile and placed on highvacuum to give crude compound 2d as a sticky solid (5.88 g, 100% yield).¹H NMR (400 MHz, CDCl₃): δ 7.21 (d, 1H, J=6.8 Hz), 6.81 (d, 1H, J=7.6Hz), 4.69-4.60 (m, 1H), 4.59-4.51 (m, 1H), 3.69 (s, 3H), 2.40-2.33 (m,2H), 2.31-2.24 (m, 2H), 1.72-1.63 (m, 4H), 1.51-1.45 (m, 3H), 1.42-1.37(m, 3H).

Step 5:

Compound 2d (5.6 g, 18.52 mmol) was dissolved in anhydrousdichloromethane (118 mL) and anhydrous methanol (58.8 mL).(5-amino-1,3-phenylene)dimethanol (2.70 g, 17.64 mmol) and EEDQ (8.72 g,35.3 mmol) were added and the reaction was stirred at room temperature,overnight. The solvent was stripped and ethyl acetate was added. Theresulting slurry was filtered, washed with ethyl acetate and dried undervacuum/N₂ to give compound 2e (2.79 g, 36% yield). ¹H NMR (400 MHz,DMSO-d6): δ 9.82 (s, 1H), 8.05, (d, 1H, J=9.2 Hz), 8.01 (d, 1H, J=7.2Hz), 7.46 (s, 2H), 6.95 (3, 1H), 5.21-5.12 (m, 2H), 4.47-4.42 (m, 4H),4.40-4.33 (m, 1H), 4.33-4.24 (m, 1H), 3.58 (s, 3H), 2.33-2.26 (m, 2H),2.16-2.09 (m, 2H), 1.54-1.46 (m, 4H), 1.30 (d, 3H, J=7.2 Hz), 1.22 (d,3H, J=4.4 Hz).

Step 6:

Compound 2e (0.52 g, 1.189 mmol) and carbon tetrabromide (1.183 g, 3.57mmol) were dissolved in anhydrous DMF (11.89 mL). Triphenylphosphine(0.935 g, 3.57 mmol) was added and the reaction stirred under argon forfour hours. The reaction mixture was diluted with DCM/MeOH (10:1) andwashed with water and brine, dried over sodium sulfate, filtered, andconcentrated. The crude material was purified by silica gelchromatography (DCM/MeOH) to give compound 2f (262 mg, 39% yield). ¹HNMR (400 MHz, DMSO-d6): δ 10.01 (s, 1H), 8.11 (d, 1H, J=6.8 Hz), 8.03(d, 1H, J=6.8 Hz), 7.67 (s, 2H), 7.21 (s, 1H), 4.70-4.64 (m, 4H),4.40-4.32 (m, 1H), 4.31-4.23 (m, 1H), 3.58 (s, 3H), 2.34-2.26 (m, 2H),2.18-2.10 (m, 2H), 1.55-1.45 (m, 4H), 1.31 (d, 3H, J=7.2 Hz), 1.21 (d,3H, J=7.2 Hz).

Step 7:

Dibromide compound 2f and IGN monomer compound I-1 were dissolved inDMF. Potassium carbonate was added and was stirred at rt ovenight. Waterwas added to the reaction mixture to precipitate the product. The slurrywas stirred at rt for 5 min and was then filtered and dried undervacuum/N₂ for 1 h. The crude material was purified by silica gelchromatography (dichloromethane/methanol) to give compound 2g (336 mg,74% yield). LCMS=5.91 min (15 min method). MS (m/z): 990.6 (M+1)⁺.

Step 8:

Diimine compound 2g was dissolved in 1,2-dichloroethane. NaBH(OAc)₃ wasadded to the reaction mixture and was stirred at rt for 1 h. Thereaction was diluted with CH₂Cl₂ and was quenched with sat'd aq NH₄Clsolution. The layers were separated and was washed with brine, driedover Na₂SO₄ and concentrated. The crude material was purified via RPHPLC(C18 column, Acetonitrile/Water) to give compound 2h (85.5 mg, 25%yield). LCMS=6.64 min (15 min method). MS (m/z): 992.6 (M+1)⁺.

Step 9:

Methylester compound 2h was dissolved in 1,2-dichloroethane.Trimethylstannanol was added to the reaction mixture and was heated at80° C. overnight. The reaction mixture was cooled to rt and was dilutedwith water. The aqueous layer was acidified to pH˜4 with 1 M HCl. Themixture was extracted with CH₂Cl₂/MeOH (10:1, 3×20 mL). The combinedorganic layers were washed with brine and was dried over Na₂SO₄ andconcentrated. The crude material was passed through a silica plug togive compound 2i (48.8 mg, 80% yield). LCMS=5.89 min (15 min method). MS(m/z): 978.6 (M+1)⁺.

Step 10:

EDC.HCl was added to a stirred solution of acid compound 2i andN-hydroxysuccinamidein CH₂Cl₂ at rt. The reaction mixture was stirredfor 2 h. The reaction mixture was diluted with CH₂Cl₂ and was washedwith water (1×15 mL) and brine (1×15 mL). The organic layer was driedover Na₂SO₄, filtered and concentrated. The crude material was purifiedvia RPHPLC (C18 column, Acetonitrile/Water) to give2,5-dioxopyrrolidin-1-yl6-(((S)-1-(((S)-1-((3-((((S)-8-methoxy-6-oxo-11,12,12a,13-tetrahydro-6H-benzo[5,6][1,4]diazepino[1,2-a]indol-9-yl)oxy)methyl)-5-((((R)-8-methoxy-6-oxo-12a,13-dihydro-6H-benzo[5,6][1,4]diazepino[1,2-a]indol-9-yl)oxy)methyl)phenyl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)amino)-6-oxohexanoate,compound D2 (8.2 mg, 30% yield). LCMS=6.64 min (15 min method). MS(m/z): 1075.4 (M+1)⁺.

Example 18 Synthesis ofN-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)-11-(3-((((S)-8-methoxy-6-oxo-11,12,12a,13-tetrahydro-6H-benzo[5,6][1,4]diazepino[1,2-a]indol-9-yl)oxy)methyl)-5-((((S)-8-methoxy-6-oxo-12a,13-dihydro-6H-benzo[5,6][1,4]diazepino[1,2-a]indol-9-yl)oxy)methyl)phenyl)-13,13-dimethyl-2,5,8-trioxa-14,15-dithia-11-azanonadecan-19-amide,Compound D6

Step 1:

To a solution of the free thiol DGN462 (40 mg, 0.042 mmol) and NHS4-(2-pyridyldithio)butanate (35 mg, 80% purity, 0.085 mmol) in anhydrousdichloromethane (0.5 mL) was added anhydrous diisopropylethylamine(0.015 mL, 0.085 mmol) and was stirred at room temperature for 16 hours.The reaction mixture was quenched with saturated ammonium chloride anddiluted with dichloromethane. The obtained mixture was separated in aseparatory funnel. The organic layer was washed with brine, dried overanhydrous sodium sulfate, filtered and stripped under reduced pressure.The residue was purified by semi-preparative reverse phase HPLC (C18column, CH₃CN/H₂O). The fractions that contained pure product werecombined, frozen and lyophilized to give the desired NHS ester, compound6a (29.7 mg, 60% yield). LCMS=9.1 min (15 min method). MS (m/z): 1157.3(M+1)⁺.

Step 2:

To a solution of the NHS ester, compound 6a (12.3 mg, 0.011 mmol) andN-(2-aminoethyl)maleimide hydrochloride (2.0 mg, 0.011 mmol) inanhydrous dichloromethane (0.3 mL) was added DIPEA (0.0022 mL, 0.013mmol). The mixture was stirred at room temperature for 3 hours then itwas stripped under reduced pressure. The residue was purified bysemi-preparative reverse phase HPLC (C18 column, CH₃CN/H₂O). Thefractions that contained pure product were combined, frozen andlyophilized to give the desired maleimide, compound D6 (10 mg, 80%yield). LCMS=8.3 min (15 min method). MS (m/z): 1181.8 (M+1)⁺.

Example 19 Synthesis ofN1-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)-N6-((S)-1-(((S)-1-((3-((((S)-8-methoxy-6-oxo-11,12,12a,13-tetrahydro-6H-benzo[5,6][1,4]diazepino[1,2-a]indol-9-yl)oxy)methyl)-5-((((S)-8-methoxy-6-oxo-12a,13-dihydro-6H-benzo[5,6][1,4]diazepino[1,2-a]indol-9-yl)oxy)methyl)phenyl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)adipamide,compound D5

NHS ester, compound 5a (8.2 mg, 7.6 μmol) and1-(2-aminoethyl)-1H-pyrrole-2,5-dione hydrochloride (2.2 mg, 0.011 mmol)were dissolved in anhydrous dichloromethane (305 μL) at roomtemperature. DIPEA (2.66 μL, 0.015 mmol) was added and the reaction andwas stirred for 3.5 hours. The reaction mixture was concentrated and waspurified by RPHPLC (C18 column, CH₃CN/H₂O, gradient, 35% to 55%). Thedesired product fractions were frozen and lyophilized to give maleimide,compound D5 as a solid white powder (5.3 mg, 58% yield). LCMS=5.11 min(8 min method). MS (m/z): 1100.6 (M+1)⁺.

Example 20 Synthesis of1-((2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)amino)-4-((5-((3-((((S)-8-methoxy-6-oxo-11,12,12a,13-tetrahydro-6H-benzo[5,6][1,4]diazepino[1,2-a]indol-9-yl)oxy)methyl)-5-((((S)-8-methoxy-6-oxo-12a,13-dihydro-6H-benzo[5,6][1,4]diazepino[1,2-a]indol-9-yl)oxy)methyl)phenyl)amino)-2-methyl-5-oxopentan-2-yl)disulfanyl)-1-oxobutane-2-sulfonicacid, compound D4

To a suspension of the free thiol, D1 (88 mg, 0.105 mmol) and1-((2,5-dioxopyrrolidin-1-yl)oxy)-1-oxo-4-(pyridin-2-yldisulfanyl)butane-2-sulfonicacid (sulfo-SPDB) (64.0 mg, 0.158 mmol) in anhydrous dichloromethane(2.10 mL) was added DIPEA (55.0 μL, 0.315 mmol) under nitrogen at roomtemperature. The mixture stirred for 16 hours and then1-(2-aminoethyl)-1H-pyrrole-2,5-dione hydrochloride (55.6 mg, 0.315mmol), anhydrous dichloromethane (1.0 mL) and DIPEA (0.055 mL, 0.315mmol) were added. The mixture stirred for an additional 5 hours at roomtemperature upon which the reaction was concentrated in vacuo. Theresulting residue was purified by RP-HPLC (C18, CH₃CN/H₂O). Fractionscontaining desired product were frozen and lyophilized to givemaleimide, D4 (20 mg, 16% yield) as a white solid. LCMS=4.92 min (8 minmethod). MS (m/z): 1158.6 (M+1)⁺.

Example 21 Synthesis ofN-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)-11-(3-((((S)-8-methoxy-6-oxo-11,12,12a,13-tetrahydro-6H-benzo[5,6][1,4]diazepino[1,2-a]indol-9-yl)oxy)methyl)-5-((((S)-8-methoxy-6-oxo-12a,13-dihydro-6H-benzo[5,6][1,4]diazepino[1,2-a]indol-9-yl)oxy)methyl)phenyl)-2,5,8-trioxa-11-azapentadecan-15-amide,compound D7

To a solution of NHS ester, 7a (5 mg, 4.82 μmol) and1-(2-aminoethyl)-1H-pyrrole-2,5-dione hydrochloride (1.7 mg, 9.64 μmol)in anhydrous dichloromethane (200 μL) was added DIPEA (1.512 μL, 8.68μmol) under nitrogen. The mixture was stirred at room temperature for 4hours and then concentrated in vacuo. The resulting residue was purifiedby RP-HPLC (C18, CH₃CN/H₂O). Fractions containing desired product werefrozen and lyophilized to give maleimide, compound D7 (3.5 mg, 68%yield). LCMS=4.61 min (15 min method). MS (m/z): 1062.8 (M+1)⁺.

Example 22 Synthesis of Compound D8

Step 1:

Tert-butyl hydroxycarbamate (1.490 g, 11.19 mmol) was dissolved inanhydrous DMF (22.38 mL). 2-(3-bromopropyl)isoindoline-1,3-dione (3 g,11.19 mmol) and potassium carbonate (3.09 g, 22.38 mmol) were added andthe reaction stirred overnight at room temperature. It was diluted withcold water and extracted with EtOAc. The organic was washed with brine,dried over sodium sulfate and the crude residue was purified by silicagel flash chromatography (EtOAc/Hex, gradient, 0% to 45%) to obtaincompound 8a as sticky solid (2.41 g, 67% yield). LCMS=4.99 min (8 minmethod). ¹H NMR (400 MHz, CDCl₃): δ 7.86-7.83 (m, 2H), 7.73-7.77 (m,2H), 7.28 (bs, 1H), 3.92 (t, 2H, J=6.0 Hz), 3.82 (t, 2H, 6.9 Hz),2.05-1.98 (m, 2H), 1.47 (s, 9H).

Step 2:

Compound 8a (2.41 g, 7.52 mmol) was dissolved in anhydrous DCM (18.81mL) and cooled to 0° C. in an ice bath. A freshly mixed solution of DCM(9.40 ml) and TFA (9.40 ml) was added and the ice bath was removed. Thereaction stirred at room temperature for 1 hour and was diluted with DCMand washed with saturated sodium bicarb. The organic layer was washedwith brine, dried, filtered and concentrated to give compound 8b (1.32g, 80% yield). The crude material was used without further purification.¹H NMR (400 MHz, CDCl₃): δ 7.85-7.82 (m, 2H), 7.72-7.69 (m, 2H), 3.78(t, 2H, J=7.0 Hz), 3.72 (t, 2H, 6.0 Hz), 1.99-1.93 (m, 2H).

Step 3:

Compound 8b (100 mg, 0.454 mmol) was dissolved in anhydrous DCM (4.5 mL)TEA (127 μl, 0.908 mmol) and 2,5-dioxopyrrolidin-1-yl(2-(trimethylsilyl)ethyl) carbonate (177 mg, 0.681 mmol) were added andthe reaction stirred at room temperature overnight. The reaction wasdiluted with DCM, washed with brine, dried, filtered, and evaporated.The crude residue was purified by silica gel flash chromatography(EtOAc/Hex, gradient, 0% to 40%) to obtain compound 8c (148 mg, 89%yield). LCMS=5.91 min (8 min method). ¹H NMR (400 MHz, CDCl₃): δ7.86-7.83 (m, 2H), 7.73-7.69 (m, 2H), 7.39 (bs, 1H), 4.26-4.20 (m, 2H),3.94 (t, 2H, J=6.0 Hz), 3.83 (t, 2H, 6.9 Hz), 2.06-1.98 (m, 2H),1.05-0.98 (m, 2H), 0.04 (s, 9H).

Step 4:

Compound 8c (148 mg, 0.406 mmol) was dissolved in Ethanol (2.7 mL) andstirred until completely soluble. Hydrazine (63.7 μl, 2.030 mmol) wasadded and the reaction stirred at room temperature until rapid formationof a white precipitate at 1 hour. The reaction was filtered throughcelite and rinsed with additional ethanol. The filtrate was evaporatedand purified by silica gel flash chromatography (A=MeOH, B=EtOAcgradient, 100% to 10%). Product fractions were detected by mass andevaporated to give compound 8d as a sticky solid (67.5 mg, 71% yield).¹H NMR (400 MHz, CDCl₃): δ 4.27-4.21 (m, 2H), 3.98 (t, 2H, J=5.9 Hz),2.92-2.87 (m, 2H), 1.85-1.77 (m, 2H), 1.06-0.99 (m, 2H), 0.04 (s, 9H).

Step 5:

Compound 2i (30 mg, 0.031 mmol) described above in Example 17 wassuspended in anhydrous DCM (613 μl). Anhydrous DMF was added dropwiseuntil the solution cleared. Compound 8d (21.57 mg, 0.092 mmol), EDC.HCl(29.4 mg, 0.153 mmol), and DMAP (0.749 mg, 6.13 μmol) were added and thereaction stirred at room temperature for 1 hour. It was diluted withDCM/MeOH 10:1 and then washed with water. The aqueous layer wasextracted with DCM/MeOH 10:1 and the combined organic was dried andconcentrated to give Compound 8e (49 mg) which was used without furtherpurification. LCMS=5.94 min (8 min method). MS (m/z): 1194.4 (M+1)⁺.

Step 6:

Compound 8e (49 mg, 0.041 mmol) was dissolved in THF (820 μl) and thereaction was cooled to 0° C. in an ice bath. TBAF (205 μl, 0.205 mmol)was added and the reaction stirred for 15 minutes before the ice bathwas removed. It was stirred at room temperature until completion. Thereaction was cooled to 0° C., quenched with saturated ammonium chlorideand extracted with DCM/MeOH 10:1. The organic was washed with brine,dried with sodium sulfate and evaporated. The crude material waspurified via RPHPLC (C18 column, Acetonitrile/Water) to give compound D8(17.6 mg, 54% yield over 2 steps). LCMS=5.1 min (8 min method). MS(m/z): 1050.4 (M+1)⁺.

Example 23 Synthesis of Compound D9

Step 1:

Compound 9a (17 mg, 0.016 mmol) was dissolved in DCM (328 μl). Compound8d (5.76 mg, 0.025 mmol) and DIPEA (5.71 μl, 0.033 mmol) were added atroom temperature and the reaction stirred until completion. It wasdiluted with 10:1 DCM:MeOH and washed with brine. The organic was driedand concentrated to give compound 9b which was used directly.

Step 2:

Compound D9 was prepared similarly as compound D8 in Example 23. Thecrude material was purified via RPHPLC (C18 column, Acetonitrile/Water)to give compound D9 (5 mg, 31% yield over 2 steps). LCMS=5.68 min (8 minmethod). MS (m/z): 1012.5 (M+1)⁺.

Example 24 In Vivo Efficacy of huCD123-CysMab-D5 inKasumi-3-Luc-mCh-Puro Disseminated Model

To test the efficacy of huCD123-CysMab-D5 for the ability to decreasedisseminated tumor burden in vivo, a luciferase-expressing disseminatedtumor model was used in combination with live animal imaging, asdescribed in the protocol below.

Female NSG mice (Jackson Labs) were each injected intravenously (IV) inthe tail vein with 5×10⁶ Kasumi-3-Luc-mCh-Puro cells, a human AML cellline engineered to express luciferase and mCherry (at Molecular Imaging,Ann Arbor, Mich.). Luciferase expression by the Kasumi-3 cells allowstumor burden to be quantified using a live animal imager, which detectsthe bioluminescence signal produced by the luciferase upon exposure invivo to the injected luciferase substrate, D-luciferin. On day 6post-inoculation, the mice were imaged and randomized into the studygroups based on bioluminescent imagining (BLI). At 24 h prior to eachadministration of conjugate, the mice were injected intraperitoneally(IP) with 400 mg/kg of non-targeted chKTI antibody to block Fc receptorson the Kasumi-3 AML cells, preventing non-specific up-take of conjugate.On days 7 and 41 post-Kasumi-3 inoculation, the mice received single IVinjections in the lateral tail vein of either vehicle, 10 μg/kg (by D5;0.80 mg/kg by huCD123) huCD123-CysMab-D5, 3 μg/kg (by D5; 0.240 mg/kg byhuCD123) huCD123-CysMab-D5 or 10 μg/kg of a non-targeted KTI-CysMab-D5control conjugate. On days 5 and 10 post-conjugate administration, themice received an IP injection of 100 mg/kg of non-targeted chKTIantibody to ensure continued blocking of Fc receptors on the AML tumorcells.

The mice were imaged on days 11, 13, 17, 20, 24, 27, 31, 38, 41, 45, 52,59, 66, 73 and 80 post-Kasumi-3 inoculation. In vivo bioluminescenceimaging was performed at Molecular Imaging (Ann Arbor, Mich.) using anIVIS 50 optical imaging (Xenogen, Alameda, Calif.). Animals were imagedthree at a time under ˜1-2% isoflurane gas anesthesia. Each mouse wasinjected IP with 150 mg/kg D-luciferin (luciferase substrate) and imagedin the prone, then supine positions, 10 minutes after the injection.Large to small binning of the CCD chip was used, and the exposure timewas adjusted (2 seconds to 2 minutes) to obtain at least several hundredcounts from the tumors that are observable in each mouse in the imageand to avoid saturation of the CCD chip. Images were analyzed usingMatlab R2015a. A custom script placed whole body fixed-volume ROIs onprone and supine images for each individual animal, and labeled based onanimal identification. Total flux (photons/sec) was calculated andexported for all ROIs to facilitate analyses between groups. The proneand supine ROIs were summed together to estimate whole body tumorburden.

% T/C was calculated as follows=[(T, median BLI of treated group)/(C,median BLI of control group)]×100%. According to NCI standards, aT/C≤42% is the minimum level of anti-tumor activity, while a T/C valueof >42% is inactive, and a T/C value of <10% is considered highlyactive.

% Tumor Burden Delay (% TBD) was calculated as follows: %TBD=(T−C)/C×100%, where T−C, where T is the time (in days) for thetreated group and C is the time (in days) for the control group, toachieve the designated BLI signal. Adapting the same metrics applied to% ILS, we consider % TBD>25 as minimally active, % TBD>40 as active, and% TBD>50 as highly active.

The mice were weighed twice a week and were monitored for clinical signsthroughout the duration of the study. Any mice reaching euthanasiacriteria were euthanized. Spontaneous deaths were recorded as they werediscovered. The study was ended on day 115 post-tumor cell inoculation.

Preliminary experiments show that treatment with either dose ofhuCD123-CysMab-D5 caused an initial regression of tumor burden overtime, reaching a nadir on day 27, while tumor burden increased steadilyin the vehicle- and KTI-CysMab-D5-treated groups during this period oftime. See, for example, FIG. 22. The tumor growth inhibition (T/C value)calculated for these preliminary experiments showed that 10 μg/kg and 3μg/kg of huCD123-CysMab-D5 are highly active at day 27, with % T/Cvalues of 0.20 and 0.25, respectively. The % Tumor Burden Delay (% TBD)was also calculated, using the BLI signal at day 45 of thevehicle-treated group as the designated BLI. According to this metric,both doses of huCD123-CysMab-D5 are highly active, resulting in % TBDof >75% (10 μg/kg huCD123-CysMab-D5) and >65% (3 μg/kghuCD123-CysMab-D5), in contrast to 0% TBD seen with 10 μg/kg of theKTI-CysMab-D5 control conjugate. In addition, treatment withhuCD123-CysMab-D5 at both 3 μg/kg and 10 μg/kg doses extended survivalin 6/6 mice with P53 mutated and multidrug resistant AML as compared tocontrol (see FIG. 23).

The survival for each of the four study groups at the end of the studyis presented in FIG. 31 and is summarized in the table below. Micetreated with vehicle had a median survival of 70 days. In contrast, micetreated with 10 μg/kg (by D5, 0.80 mg/kg by huCD123) ofhuCD123-CysMab-D5 had a median survival of 115 days, resulting in a 64%ILS (highly active). Likewise, mice treated with 3 μg/kg (by D5, 0.240mg/kg by huCD123) of huCD123-CysMab-D5 had a median survival time of 105days, resulting in a 50% ILS (highly active). Mice treated with 10 μg/kg(by D5) of huKTI-CysMab-D5 non-targeted control conjugate had a mediansurvival of 65.5 days, which generated a 0% ILS (inactive), indicatingthe high activity obtained with huCD123-CysMab-D5 is CD123-dependent.

Dose Median Dose (mg/kg Survival Group Treatment (μg/kg D5) huCD123)(Days) T-C % ILS Activity 1 Vehicle — — 70 — — — 2 huCD123- 10 0.80 11545 64 Highly CysMab-D5 Active 3 huCD123- 3 0.240 105 35 50 HighlyCysMab-D5 Active 4 huKTI- 10 0.80 65.5 0 0 Inactive CysMab-D5For N=6, the media survival (days) is the mean of the days when the3^(rd) and 4^(th) mice are lost. Using the median survival values, the %ILS (Increased Life Span) is calculated as: % ILS=(T−C)/C×100%, where Tis the media survival (in days) of the treated group and C is the mediansurvival (in days) of the control group. NCI standards for disseminatedmodels are: ILS≥25% is minimally active, ILS>40% is active, and ILS≥50%is highly active.

Example 25 huCD123-CysMab-D5 Conjugate Induces DNA Damage Leading CellCycle Arrest in S-Phase and Apoptosis-Mediated Cell Death of MV4-11Cells

To evaluate the mechanism of the huCD123-CysMab-D5-mediated cell death,CD123-expressing MV4-11 AML cells were treated with 10 nM ofhuCD123-CysMab-D5 for one hour, followed by an additional incubation ina conjugate-free culture medium for 48 hours at 37° C. Untreated MV4-11cells were used as a control. The cells were harvested and stained withvarious reagents to quantify the number of cells in different stages ofthe cell cycle (propidium iodide), cells with DNA damage (pH2AX),apoptosis (Annexin-V and cleaved Caspase-3) and perforated plasmamembrane (TO-PRO-3). As demonstrated in FIG. 24, incubation of MV4-11cells with huCD123-CysMab-D5 leads to DNA damage, arrest in S-phase ofthe cell cycle, and apoptosis-mediated cell death.

Example 26 In Vivo Efficacy of huCD123-CysMab-D5 in Molm-13 DisseminatedModel

Data collection and analysis for all disseminated models: The mice wereweighed twice a week and were monitored for clinical signs throughoutthe duration of the study. The measured end-point was survival. Animalswere euthanized when hind leg paralysis was present, body weightdecreased by >20% of pre-treatment weight, a visible tumor appeared, orany signs of distress were visible. Spontaneous deaths were recordedwhen they were discovered. For disseminated models, Tumor Growth Delayis calculated as T−C, where T is the median survival time (in days) of atreated group and C is the median survival time (in days) of the vehiclecontrol group. The Percent Increased Life Span (% ILS) for disseminatedmodels is calculated using the following formula: % ILS=(T−C)/C×100%.Anti-tumor activity was evaluated as per NCI standards for disseminatedmodels: ILS≥25% is minimum active, ILS>40% is active, and ILS≥50% ishighly active.

To test the efficacy of huCD123-CysMab-D5 for the ability to decreasetumor burden in vivo, a disseminated tumor model was used as describedin the protocol below.

Female athymic nude mice were each injected intravenously in the lateraltail vein with 10×10⁶ Molm-13 cells, a human AML cell line, in 100 μl ofserum-free medium. On day 7 post-inoculation, mice were randomized intothe study groups. At 24 h prior to conjugate administration, the micewere injected intraperitoneally with 400 mg/kg of non-targeted chKTIantibody to block Fc receptors on the Molm-13 AML cells, preventingnon-specific up-take of conjugate. On day 7 post-Molm-13 inoculation,the mice received a single intravenous injection, in the lateral tailvein, of vehicle, 0.1 μg/kg (by D5; 0.008 mg/kg by huCD123)huCD123-CysMab-D5, 0.3 μg/kg (by D5; 0.024 mg/kg by huCD123)huCD123-CysMab-D5, 1 μg/kg (by D5; 0.08 mg/kg by huCD123)huCD123-CysMab-D5, 1 μg/kg (by D5; 0.08 mg/kg by huKTI) huKTI-CysMab-D5control conjugate, 0.1 μg/kg (by D2; 0.0059 mg/kg by huCD123)huCD123-lysine linked-D2, 0.3 μg/kg (by D2; 0.018 mg/kg by huCD123)huCD123-lysine linked-D2, 1 μg/kg (by D2; 0.059 mg/kg by huCD123)huCD123-lysine linked-D2 or 1 μg/kg (by D2; 0.059 mg/kg by chKTI)chKTI-lysine linked-D2 control conjugate. On days 4 and 9 post-conjugateadministration, the mice received intraperitoneal injections of 100mg/kg of non-targeted chKTI antibody to ensure continued blocking of Fcreceptors on the AML tumor cells. The results are summarized in thetable below and in FIG. 25.

The huCD123-CysMab-D5 conjugate was highly active at all three dosestested, each generating a % ILS of >262.5 days. In contrast, a 1 μg/kg(by D5) dose of non-targeted huKTI-CysMab-D5 control conjugate wasinactive, generating a 0% ILS. This demonstrates the CD123-dependentactivity of huCD123-CysMab-D5. Similarly, huCD123-lysine linked-D2 washighly active at all three doses tested, each generating a % ILS of >59.However, a 1 μg/kg (by D2) dose of chKTI-lysine linked-D2 non-targetedcontrol conjugate was also inactive, generating a 11% ILS, demonstratingthe CD123-dependent activity of huCD123-lysine linked-D2. The oneobvious difference between huCD123-CysMab-D5 and huCD123-lysinelinked-D2 can be seen when comparing the % ILS obtained with the 0.1μg/kg dose, the lowest dose tested, of each CD123-targeted conjugate.The % ILS obtained with 0.1 μg/kg of huCD123-CysMab-D5 was 262.5 days,while that obtained with 0.1 μg/kg dose of huCD123-lysine linked-D2 was59 days, pointing to the superiority of huCD123-CysMab-D5 in this model.

Tumor Median Growth Survival Delay Time (T-C, Treatment Group (Days)Days) % ILS Result Vehicle 28 0 0 — huCD123-CysMab-D5 101.5 73.5 262.5Highly (0.1 μg/kg) Active huCD123-CysMab-D5 >101.5 >73.5 >262.5 Highly(0.3 μg/kg) Active huCD123-CysMab-D5 >101.5 >73.5 >262.5 Highly (1μg/kg) Active huKTI-CysMab-D5 26.5 0 0 Inactive (1 μg/kg) huCD123-lysinelinked-D2 44.5 16.5 59 Highly (0.1 μg/kg) Active huCD123-lysinelinked-D2 >101.5 >73.5 >262.5 Highly (0.3 μg/kg) Active huCD123-lysinelinked-D2 >101.5 >73.5 >262.5 Highly (1 μg/kg) Active chKTI-lysinelinked-D2 31 3 11 Inactive (1 μg/kg)

Example 27 In Vivo Efficacy of huCD123-CysMab-D5 in EOL-1 SubcutaneousModels

Data collection and analysis for all subcutaneous models: The mice wereweighed twice a week and were monitored for clinical signs throughoutthe duration of the study. Animals were euthanized when hind legparalysis was present, body weight decreased by >20% of pre-treatmentweight, tumor ulceration occurred, or when any signs of distress werevisible. Tumor volumes were measured one to two times weekly in threedimensions using a caliper. The tumor volume was expressed in mm³ usingthe formula V=Length×Width×Height×½ (Tomayko and Reynolds, CancerChemother. Pharmacol. 24: 148-54 (1989)). Activity was assessed asdescribed in Bissery et al., Cancer Res. 51: 4845-52 (1991). TumorGrowth Inhibition (T/C Value) was also assessed using the followingformula: T/C (%)=(Median tumor volume of the treated/Median tumor volumeof the control)×100%. Tumor volume was determined simultaneously for thetreated (T) and the vehicle control (C) groups when tumor volume of thevehicle control reached a predetermined size (Bissery et al., CancerRes. 51: 4845-52 (1991). The daily median tumor volume of each treatedgroup was determined, including tumor-free mice (0 mm³). According toNCI standards, a T/C≤42% is the minimum level of anti-tumor activity. AT/C<10% is considered a high anti-tumor activity level.

To test the efficacy of huCD123-CysMab-D5 for the ability to decreasetumor burden in vivo, three studies on a subcutaneous tumor model wasused as described in the protocols below.

In a first study, Female athymic nude mice were each inoculated with10×10⁶ EOL-1 cells, a human AML cell line, in 100 μl serum freemedium/matrigel subcutaneously in the right flank. On day 6 post-EOL-1inoculation, mice were randomized into the study groups. At 24 h priorto conjugate administration, the mice were injected intraperitoneallywith 400 mg/kg of non-targeted chKTI antibody to block Fc receptors onthe EOL-1 AML cells, preventing non-specific up-take of conjugate. Onday 7 post-EOL-1 inoculation, the mice received a single intravenousinjection, in the lateral tail vein, of vehicle, 1 μg/kg (by D5; 0.08mg/kg by huCD123) huCD123-CysMab-D5, 3 μg/kg (by D5; 0.24 mg/kg byhuCD123) huCD123-CysMab-D5, 1 μg/kg (by D2; 0.050 mg/kg by huCD123)huCD123-lysine linked-D2, 3 μg/kg (by D2, 0.151 mg/kg by huCD123)huCD123-lysine linked-D2, 1 μg/kg (by D5; 0.08 mg/kg by huKTI)huKTI-CysMab-D5 control conjugate, 3 μg/kg (by D5; 0.24 mg/kg by huKTI)huKTI-CysMab-D5 control conjugate, 1 μg/kg (by D2; 0.050 mg/kg by chKTI)chKTI-lysine linked-D2 or 3 μg/kg (by D2; 0.151 mg/kg by chKTI)chKTI-lysine linked-D2 control conjugate. On days 4 and 9 post-conjugateadministration, the mice received an intraperitoneal injection of 100mg/kg of non-targeted chKTI antibody to ensure continued blocking of Fcreceptors on the AML tumor cells. The results are represented in thetable below and in FIG. 26.

The 1 μg/kg (by D5) and the 3 μg/kg doses of huCD123-CysMab-D5 wereactive and highly active, respectively, generating 13% (3/6 CRs) and 2%T/C (5/6 CRs), respectively. In contrast, the 1 μg/kg (by D5) and the 3μg/kg doses of huKTI-CysMab-D5 non-targeted control conjugate wereinactive, with % T/C of >73, demonstrating that the activity ofhuCD123-CysMab-D5 was CD123-dependent. The 1 μg/kg (by D2) and the 3μg/kg doses of huCD123-lysine linked-D2 were active and highly active,respectively, generating 30% (1/6 CRs) and 1% (6/6 CRs), respectively.In contrast, the 1 μg/kg (by D2) and the 3 μg/kg doses of thechKTI-lysine linked-D2 non-targeted control conjugate were bothinactive, generating a 75% T/C (0/6 CRs) and an 81% T/C (0/6 CRs),respectively. This demonstrates the activity of huCD123-lysine linked-D2was CD123-dependent. A difference between the two CD123-targetingconjugates becomes apparent when comparing 1 μg/kg (by D5) ofhuCD123-CysMab-D5 with 1 μg/kg (by D2) of huCD123-lysine linked-D2, inthat the former results in a 13% T/C and 3/6 CRs and the latter resultsin a 30% T/C and only 1/6 CR, demonstrating the apparent superiority ofhuCD123-CysMab-D5 in this model.

% T/C Treatment Group (Day 15) PR CR Result Vehicle — 1/6 1/6 —huCD123-CysMab-D5 (1 μg/kg) 13 3/6 3/6 Active huCD123-CysMab-D5 (3μg/kg) 2 5/6 5/6 Highly Active huCD123-lysine linked-D2 (1 μg/kg) 30 1/61/6 Active huCD123-lysine linked-D2 (3 μg/kg) 1 6/6 6/6 Highly ActivehuKTI-CysMab-D5 (1 μg/kg) 100 0/6 0/6 Inactive huKTI-CysMab-D5 (3 μg/kg)73 1/6 1/6 Inactive chKTI-lysine linked-D2 (1 μg/kg) 75 0/6 0/6 InactivechKTI-lysine linked-D2 (3 μg/kg) 81 0/6 0/6 Inactive

In a second study, female athymic nude mice were each inoculated with10×10⁶ EOL-1 cells, a human AML cell line, in 100 μl serum freemedium/matrigel subcutaneously in the right flank. On day 6 post-EOL-1inoculation, mice were randomized into the study groups. At 24 h priorto conjugate administration, the mice were injected intraperitoneallywith 400 mg/kg of non-targeted chKTI antibody to block Fc receptors onthe EOL-1 AML cells, preventing non-specific up-take of conjugate. Onday 7 post-EOL-1 inoculation, the mice received a single intravenousinjection, in the lateral tail vein, of vehicle, 0.5 μg/kg (by D5; 0.04mg/kg by huCD123) huCD123-CysMab-D5 or 1 μg/kg (by D5; 0.08 mg/kg byhuCD123) huCD123-CysMab-D5. On days 4 and 9 post-conjugateadministration, the mice received an intraperitoneal injection of 100mg/kg of non-targeted chKTI antibody to ensure continued blocking of Fcreceptors on the AML tumor cells. The results are represented in thetable below and in FIG. 27.

The 0.5 μg/kg (by D5) dose of huCD123-CysMab-D5 was active, generatingan 11% T/C and 3/6 CRs. Similarly, the 1 μg/kg dose of huCD123-CysMab-D5was highly active, generating a 3% T/C and 6/6 CRs.

% T/C Treatment Group (Day 16) PR CR Result Vehicle — 0/6 0/6 —huCD123-CysMab-D5 (0.5 μg/kg) 11 3/6 3/6 Active huCD123-CysMab-D5 (1μg/kg) 3 6/6 6/6 Highly Active

In a third study, female athymic nude mice were each inoculated with10×10⁶ EOL-1 cells, a human AML cell line, in 100 μl serum freemedium/matrigel subcutaneously in the right flank. On day 6 post-EOL-1inoculation, mice were randomized into the study groups. At 24 h priorto conjugate administration, the mice were injected intraperitoneallywith 400 mg/kg of non-targeted chKTI antibody to block Fc receptors onthe EOL-1 AML cells, preventing non-specific up-take of conjugate. Onday 7 post-EOL-1 inoculation, mice received a single intravenousinjection, in the lateral tail vein, of vehicle, 3 μg/kg (by D5; 0.24mg/kg by huCD123) huCD123-CysMab-D5, 3 μg/kg (by D5) FGN849 (free drugform of the payload), 0.24 mg/kg unconjugated (naked) huCD123 antibody,or 3 μg/kg (by D5; 0.24 mg/kg by huKTI) huKTI-CysMab-D5 controlconjugate. Mice treated with Cytarabine received a singleintraperitoneal injection on days 7, 8, 9, 10 and 11 post-EOL-1inoculation, and mice treated with Azacitidine received a singleintraperitoneal injection on days 7, 10, 13, 16 and 19 post EOL-1inoculation. On days 4 and 9 post-conjugate administration, the micereceived an intraperitoneal injection of 100 mg/kg of non-targeted chKTIantibody to ensure continued blocking of Fc receptors on the AML tumorcells. The results are represented in the table below and in FIG. 28.

Out of all the articles tested, only the 3 μg/kg (by D5) dose ofhuCD123-CysMab-D5 demonstrated activity, generating a highly active 1%T/C and 8/8 CRs, in contrast to the 3 μg/kg (by D5) dose ofhuKTI-CysMab-D5 non-targeting control conjugate, which generated a 69%T/C and 0/8 CRs. A 3 μg/kg (by D5) dose of the unconjugated free drug,FGN849, was also inactive, with a 92% T/C and 0/8 CRs. Likewise, a 0.24mg/kg dose of unconjugated (“naked”) huCD123 antibody, matching theantibody dose of huCD123-CysMab-D5, was inactive, with a 60% T/C and 0/8CRs. Cytarabine, administered at a dose of 75 mg/kg daily for 5 days,was inactive, with an 84% T/C and 0/8 CRs. Azacitidine, administered ata dose of 3.75 mg/kg once every three days for 5 doses, was inactive,with a 59% T/C and 0/8 CRs.

% T/C Treatment Group (Day 16) PR CR Result Vehicle — 0/8 0/8 —huCD123-CysMab-D5 (3 μg/kg) 1 8/8 8/8 Highly Active FGN849 (3 μg/kg) 920/8 0/8 Inactive Naked antibody (0.24 mg/kg) 60 0/8 0/8 InactivehuKTI-CysMab-D5 (3 μg/kg) 69 0/8 0/8 Inactive Cytarabine; 75 mg/kg, qd ×5 84 0/8 0/8 Inactive Azacitidine; 3.75 mg/kg, q3d × 5 59 0/8 0/8Inactive

Example 28 In Vivo Efficacy of huCD123-CysMab-D5 and huCD123-SeriMab-sD1in MV4-11 Disseminated Model

To test the efficacy of huCD123-CysMab-D5 and huCD123-SeriMab-sD1 forthe ability to decrease tumor burden in vivo, a disseminated tumor modelwas used as described in the protocol below.

Female NOD SCID mice were pre-treated with 150 mg/kg cyclophosphamide topartially ablate bone marrow in order to improve the engraftment ofMV4-11 cells. The cyclophosphamide (Baxter, Lot #4E011, Exp May 2017)was reconstituted with 0.9% NaCl and was administered intraperitoneallyto the mice on day −3 and day −2 prior to MV4-11 cell inoculation on day0. Following cyclophosphamide treatment as described above, the micewere each injected intravenously in the lateral tail vein with 3×10⁶MV4-11 cells, a human AML cell line, in 100 μl of serum-free medium. Onday 7 post-MV4-11 inoculation, mice were randomized into the studygroups. At 24 h prior to conjugate administration, the mice wereinjected intraperitoneally with 400 mg/kg of non-targeted chKTI antibodyto block Fc receptors on the MV4-11 AML cells, preventing non-specificup-take of conjugate. On day 7 post-MV4-11 inoculation, the micereceived a single intravenous injection, in the lateral tail vein, ofvehicle, 1 μg/kg (by D5; 0.08 mg/kg by huCD123) huCD123-CysMab-D5, 3μg/kg (by D5; 0.24 mg/kg huCD123) huCD123-CysMab-D5, 1 μg/kg (by D1;0.054 mg/kg by huCD123) huCD123-SeriMab-sD1, 3 μg/kg (by D1; 0.163 mg/kgby huCD123) huCD123-SeriMab-sD1, 1 μg/kg (by D5; 0.08 mg/kg by huKTI)huKTI-CysMab-D5 control conjugate, 3 μg/kg (by D5; 0.24 mg/kg by huKTI)huKTI-CysMab-D5 control conjugate, 1 μg/kg (by D1; 0.07 mg/kg by chKTI)chKTI-SeriMab-sD1 control conjugate or 3 μg/kg (by D1; 0.21 mg/kg bychKTI) chKTI-SeriMab-sD1 control conjugate. The results are summarizedin the table below and in FIG. 29.

Both the 1 μg/kg and the 3 μg/kg (by D5) doses of huCD123-CysMab-D5 werehighly active, each generating a % ILS of ≥70. In contrast, the 1 μg/kgdose (by D5) of huKTI-CysMab-D5 non-targeted control conjugate wasminimally active, generating a 28% ILS. The 3 μg/kg dose ofhuKTI-CysMab-D5 was inactive, with a 0% ILS, demonstrating theCD123-dependent activity of huCD123-CysMab-D5. The 1 μg/kg and the 3μg/kg (by sD1) doses of huCD123-SeriMab-sD1 were both highly active,each generating a % ILS of ≥101. However, when the activity of thenon-targeted chKTI-SeriMab-sD1 control conjugates is examined, a highdegree of non-specific, non-CD123-target activity is apparent from the 3μg/kg dose (by sD1), which generates a 65% ILS (highly active). Thisindicates some of the high activity of the 3 μg/kg (by sD1) dose ofCD123-targeting huCD123-SeriMab-sD1 is likely due to non-specific drugup-take mechanisms that do not involve targeting CD123. In contrast, the1 μg/kg (by sD1) dose of chKTI-SeriMab-sD1 control conjugate isinactive, with a % ILS of 15, demonstrating that the non-specificactivity of the 3 μg/kg dose of chKTI-SeriMab-sD1 is dose-dependent andthat the high activity of the 1 μg/kg (by sD1) dose ofhuCD123-SeriMab-sD1 is indeed CD123-dependent.

Tumor Median Growth Survival Delay Time (T-C, Treatment Group (Days)Days) % ILS Result Vehicle 46 0 0 — huCD123-CysMab-D5 81 35 76 HighlyActive (1 μg/kg) huCD123-CysMab-D5 78 32 70 Highly Active (3 μg/kg)huCD123-SeriMab-sD1 92.5 46.5 101 Highly Active (1 μg/kg)huCD123-SeriMab-sD1 >92.5 >46.5 >101 Highly Active (3 μg/kg)huKTI-CysMab-D5 59 13 28 Minimally Active (1 μg/kg) huKTI-CysMab-D5 46 00 Inactive (3 μg/kg) chKTI-SeriMab-sD1 53 7 15 Inactive (1 μg/kg)chKTI-SeriMab-sD1 76 30 65 Highly Active (3 μg/kg)

Example 29 In Vivo Efficacy of huCD123-CysMab-D5 and huCD123-SeriMab-sD1in MV4-11 Subcutaneous Model

To test the efficacy of huCD123-CysMab-D5 and huCD123-SeriMab-sD1 forthe ability to decrease tumor burden in vivo, a subcutaneous tumor modelwas used as described in the protocol below.

Female CB.17 SCID mice were each inoculated with 10×10⁶ MV4-11 cells, ahuman AML cell line, in 100 μl serum free medium/matrigel subcutaneouslyin the right flank. On day 14 post-MV4-11 inoculation, mice wererandomized into the study groups. At 24 h prior to conjugateadministration, the mice were injected intraperitoneally with 400 mg/kgof non-targeted chKTI antibody to block Fc receptors on the MV4-11 AMLcells, preventing non-specific up-take of conjugate. On day 15post-MV4-11 inoculation, the mice received a single intravenousinjection, in the lateral tail vein, of vehicle, 0.3 μg/kg (by D1; 0.016mg/kg by huCD123) huCD123-SeriMab-sD1, 1 μg/kg (by D1; 0.054 mg/kg byhuCD123) huCD123-SeriMab-sD1, 3 μg/kg (by D1; 0.16 mg/kg by huCD123)huCD123-SeriMab-sD1, 0.3 μg/kg (by D5; 0.024 mg/kg by huCD123)huCD123-CysMab-D5, 1 μg/kg (by D5; 0.08 mg/kg by huCD123)huCD123-CysMab-D5, 3 μg/kg (by D5; 0.24 mg/kg by huCD123)huCD123-CysMab-D5, 0.3 μg/kg (by D1; 0.021 mg/kg by chKTI)chKTI-SeriMab-sD1 control conjugate, 1 μg/kg (by D1; 0.07 mg/kg bychKTI) chKTI-SeriMab-sD1 control conjugate, 3 μg/kg (by D1; 0.21 mg/kgby chKTI) chKTI-SeriMab-sD1 control conjugate, 0.3 μg/kg (by D5; 0.024mg/kg by huKTI) huKTI-CysMab-D5 control conjugate, 1 μg/kg (by D5; 0.08mg/kg by huKTI) huKTI-CysMab-D5 control conjugate or 3 μg/kg (by D5;0.24 mg/kg by huKTI) huKTI-CysMab-D5 control conjugate. On day 20post-MV4-11 inoculation, the mice injected intraperitoneally with 100mg/kg of non-targeted chKTI antibody to ensure continued blocking of Fcreceptors on the AML tumor cells. The results are represented in thetable below and in FIG. 30.

The 1 μg/kg and the 3 μg/kg (by sD1) doses of huCD123-SeriMab-sD1 wereboth highly active, each generating a % T/C of 0 and 6/6 CRs. The lowesthuCD123-SeriMab-sD1 dose tested, 0.3 μg/kg (by sD1) was inactive, with a% T/C of 43 and 0/6 CRs. However, when the activity of the non-targetedchKTI-SeriMab-sD1 control conjugates is examined, a high degree ofnon-specific, non-CD123-target activity is apparent from the 3 μg/kgdose (by sD1), which generates a 6% T/C (highly active) and 3/6 CRs.This indicates some of the high activity of the 3 μg/kg (by sD1) dose ofCD123-targeting huCD123-SeriMab-sD1 is likely due to non-specific drugup-take mechanisms that do not involve targeting CD123. In contrast,both the 1 μg/kg (by sD1) and the 0.3 μg/kg doses of chKTI-SeriMab-sD1control conjugate were inactive, generating 73% T/C and 83% T/C,respectively, demonstrating that the high non-specific activity of the 3μg/kg dose (by sD1) of chKTI-SeriMab-sD1 is dose-dependent and that thehigh activity of the 1 μg/kg (by sD1) dose of huCD123-SeriMab-sD1 isindeed CD123-dependent.

The 1 μg/kg and the 3 μg/kg (by D5) doses of huCD123-CysMab-D5 were bothhighly active, each generating a 0% T/C, and 5/6 and 6/6 CRs,respectively. The 0.3 μg/kg (by D5) dose of huCD123-CysMab-D5, thelowest dose tested, was inactive, with a 69% TIC and 0/6 CRs. Incontrast, all three doses (by D5) of the huKTI-CysMab-D5 non-targetedcontrol conjugate were inactive, each generating ≥43% TIC and 0/6 CRs;demonstrating the CD123-dependent activity of huCD123-CysMab-D5.

% T/C Treatment Group (Day 49) PR CR Result Vehicle — 0/6 0/6 —huCD123-SeriMab-sD1 (0.3 μg/kg) 43 1/6 0/6 Inactive huCD123-SeriMab-sD1(1 μg/kg) 0 6/6 6/6 Highly Active huCD123-SeriMab-sD1 (3 μg/kg) 0 6/66/6 Highly Active huCD123-CysMab-D5 (0.3 μg/kg) 69 0/6 0/6 InactivehuCD123-CysMab-D5 (1 μg/kg) 0 5/6 5/6 Highly Active huCD123-CysMab-D5 (3μg/kg) 0 6/6 6/6 Highly Active chKTI-SeriMab-sD1 (0.3 μg/kg) 83 0/6 0/6Inactive chKTI-SeriMab-sD1 (1 μg/kg) 73 0/6 0/6 InactivechKTI-SeriMab-sD1 (3 μg/kg) 6 5/6 3/6 Highly Active huKTI-CysMab-D5 (0.3μg/kg) 102 0/6 0/6 Inactive huKTI-CysMab-D5 (1 μg/kg) 79 0/6 0/6Inactive huKTI-CysMab-D5 (3 μg/kg) 43 0/6 0/6 Inactive

Example 30 In Vivo Tolerability of huCD123-IGN Conjugates in Mice

To test the tolerability of huCD123-CysMab-D5 and other huCD123-IGNconjugates in vivo, a mouse model was used as described in the protocolbelow.

Female CD-1 mice received a single intravenous injection into thelateral tail vein of vehicle, 150 μg/kg (by D5, 12 mg/kg by huCD123) ofhuCD123-CysMab-D5, 125 μg/kg (by D5, 10 mg/kg by huCD123) ofhuCD123-CysMab-D5, 100 μg/kg (by D5, 8 mg/kg by huCD123) ofhuCD123-CysMab-D5, 150 μg/kg (by sD1, 14.3 mg/kg by huCD123) ofhuCD123-SeriMab-sD1 or 125 μg/kg (by sD1, 11.9 by huCD123) ofhuCD123-SeriMab-sD1. The huCD123 antibody does not cross-react withmouse CD123, which makes this in vivo mouse model an indicator ofoff-target toxicity only. The mice were observed daily for 33 days, andbody weights were determined. If an animal experienced greater than 20%body weight loss or became moribund, the animal was euthanized. Otherthan body weight loss, no other clinical observations were made duringthe course of the study in any of the treatment.

Female CD-1 mice received a single intravenous injection into thelateral tail vein of vehicle, 75 μg/kg (by D2, 4.4 mg/kg by huCD123) ofhuCD123-lysine linked-D2, 100 μg/kg (by D2, 5.9 mg/kg by huCD123) ofhuCD123-lysine linked-D2 or 125 μg/kg (by D2, 7.4 mg/kg by huCD123) ofhuCD123-lysine linked-D2. The huCD123 antibody does not cross-react withmouse CD123, which makes this in vivo mouse model an indicator ofoff-target toxicity only. The mice were observed daily for 22 days, andbody weights were determined. If an animal experienced greater than 20%body weight loss or became moribund, the animal was euthanized.

The results are summarized in the tables below and in FIG. 32 and FIG.33.

huCD123-CysMab-D5 was not tolerated at 150 μg/kg (by D5, 12 mg/kg byhuCD123). The nadir of mean change in body weight occurred on day 12,with an 11% decrease. One out of eight mice was euthanized on day 12 dueto >20% body weight loss. huCD123-CysMab-D5 was not well tolerated at125 μg/kg (by D5, 10 mg/kg by huCD123). The nadir of mean change in bodyweight occurred on day 10, with an 8.6% decrease. One out of eight micewas euthanized on day 9 due to body weight loss. huCD123-CysMab-D5 wastolerated at 100 μg/kg (by D5, 8 mg/kg by huCD123). The nadir of meanchange in body weight occurred on day 6, with a 7% decrease. None of themice in this treatment group were euthanized due to body weight loss.

huCD123-SeriMab-sD1 was not tolerated at 150 μg/kg (14.3 mg/kg byhuCD123). The nadir of mean change in body weight occurred on day 10,with a 17.3% decrease. Two out of eight mice were euthanized on day 10due to body weight loss. huCD123-SeriMab-sD1 was not well tolerated at125 μg/kg (by sD1, 11.9 mg/kg by huCD123). The nadir of mean change inbody weight occurred on day 10, with a 6.7% decrease. One out of eightmice was euthanized on day 12 due to body weight loss.

huCD123-lysine linked-D2 was tolerated at 75 μg/kg (by D2, 4.4 mg/kg byhuCD123). The nadir of mean change in body weight occurred on day 5,with a 6% decrease. None of the mice in this treatment group wereeuthanized due to body weight loss. huCD133-lysine linked-D2 wastolerated at 100 μg/kg (by D2, 5.9 mg/kg by huCD123). The nadir of meanchange in body weight occurred on day 7, with an 8% decrease. None ofthe mice in this treatment group were euthanized due to body weightloss. huCD123-lysine linked-D2 was not tolerated at 125 μg/kg (by D2,7.4 mg/kg by huCD123). The nadir of mean change in body weight occurredon day 9 (when N=6), with a 17% decrease. The following numbers of mice,out of the original eight, were euthanized on the days indicated, dueto >20% body weight loss: one on day 8, one on day 9, two on day 10, oneon day 11 and one on day 13.

% Number decrease of mice in μg/kg euthanized Body mean by mg/kg forweight body drug by >20 BW nadir weight Treatment payload huCD123 loss(day) at nadir Vehicle — — 0/8 — — huCD123-CysMab-D5 150 12 1/8 12 11huCD123-CysMab-D5 125 10 1/8 10 8.6 huCD123-CysMab-D5 100 8 0/8 6 7huCD123-SeriMab- 150 14.3 2/8 10 17.3 sD1 huCD123-SeriMab- 125 11.9 1/810 6.7 sD1 Vehicle — — 0/8 — — huCD123-lysine 75 4.4 0/8 5 6 linked-D2huCD123-lysine 100 5.9 0/8 7 8 linked-D2 huCD123-lysine 125 7.4 6/8 9 17linked-D2 BW: Body weight

We claim:
 1. An antibody or antigen-binding fragment thereof that bindsto human CD123 or human IL-3Rα, said antibody or antibody fragmentthereof, comprising: a) a heavy chain variable region CDR1 having theamino acid sequence selected from the group consisting of SEQ ID NOs: 1,5, and 12; a heavy chain variable region CDR2 having the amino acidsequence selected from the group consisting of SEQ ID NOs: 2, 3, 6-10,13, and 14; and a heavy chain variable region CDR3 having the amino acidsequence selected from the group consisting of: SEQ ID NOs: 4, 11, and15; and b) a light chain variable region CDR1 having the amino acidsequence selected from the group consisting of: SEQ ID NOs: 16, 19, 20,23 and 72; a light chain variable region CDR2 having the amino acidsequence selected from the group consisting of: SEQ ID NOs: 17, 21, 24and 71; and a light chain variable region CDR3 having the amino acidsequence selected from the group consisting of: SEQ ID NOs: 18, 22, and25; wherein the antibody or antigen-binding fragment thereof binds anepitope within amino acids 101 to 346 of human CD123 or IL3-Rα antigenand inhibits IL3-dependent proliferation in antigen-positive TF-1 cells.2. The antibody or antigen-binding fragment thereof of claim 1,comprising: a) an immunoglobulin heavy chain variable region having theamino acid sequence set forth in SEQ ID NO: 34; and b) an immunoglobulinlight chain variable region having the amino acid sequence set forth inSEQ ID NO:
 35. 3. The antibody or antigen-binding fragment thereof ofclaim 2, wherein Xaa, the second residue from the N-terminus of SEQ IDNO: 34, is Phe.
 4. The antibody or antigen-binding fragment thereof ofclaim 2, wherein Xaa, the second residue from the N-terminus of SEQ IDNO: 34, is Val.
 5. A polypeptide comprising the heavy chain variableregion and the light chain variable region sequences of claim
 2. 6. Theantibody or antigen-binding fragment thereof of claim 1, comprising: a)an immunoglobulin heavy chain variable region having the amino acidsequence set forth in SEQ ID NO: 34; and b) an immunoglobulin lightchain variable region having the amino acid sequence set forth in SEQ IDNO:
 37. 7. The antibody or antigen-binding fragment thereof of claim 1,comprising: a) an immunoglobulin heavy chain region having the aminoacid sequence set forth in SEQ ID NO: 54; and b) an immunoglobulin lightchain variable region having the amino acid sequence set forth in SEQ IDNO:
 35. 8. The antibody or antigen-binding fragment thereof of claim 7,wherein Xaa, the second residue from the N-terminus of SEQ ID NO: 54, isPhe.
 9. The antibody or antigen-binding fragment thereof of claim 7,wherein Xaa, the second residue from the N-terminus of SEQ ID NO: 54, isVal.
 10. The antibody or antigen-binding fragment thereof of claim 1,comprising: a) a heavy chain variable region CDR1 having the amino acidsequence set forth in SEQ ID NO: 5; a heavy chain variable region CDR2having the amino acid sequence set forth in SEQ ID NO: 6, 7, 8, 9, or10; and a heavy chain variable region CDR3 having the amino acidsequence set forth in SEQ ID NO: 11; and b) a light chain variableregion CDR1 having the amino acid sequence set forth in SEQ ID NO: 19 20or 72; a light chain variable region CDR2 having the amino acid sequenceset forth in SEQ ID NO: 21 or 71; and a light chain variable region CDR3having the amino acid sequence set forth in SEQ ID NO:
 22. 11. A cellproducing the antibody or antigen-binding fragment thereof of claim 1.12. A method of producing the antibody or antigen-binding fragmentthereof of claim 1, comprising: (a) culturing a cell producing saidantibody or antigen-binding fragment thereof; and, (b) isolating saidantibody, antigen-binding fragment thereof, or polypeptide from saidcultured cell.
 13. A pharmaceutical composition comprising the antibodyor antigen-binding fragment thereof of claim 1, and a pharmaceuticallyacceptable carrier.
 14. The antibody or antigen-binding fragment thereofof claim 1, comprising: a) a heavy chain variable region CDR1 having theamino acid sequence set forth in SEQ ID NO: 1; a heavy chain variableregion CDR2 having the amino acid sequence set forth in SEQ ID NO: 2 or3; and a heavy chain variable region CDR3 having the amino acid sequenceset forth in SEQ ID NO: 4; and b) a light chain variable region CDR1having the amino acid sequence set forth in SEQ ID NO: 16; a light chainvariable region CDR2 having the amino acid sequence set forth in SEQ IDNO: 17; and a light chain variable region CDR3 having the amino acidsequence set forth in SEQ ID NO:
 18. 15. The antibody or antigen-bindingfragment thereof of claim 1, comprising: a) a heavy chain variableregion CDR1 having the amino acid sequence set forth in SEQ ID NO: 12; aheavy chain variable region CDR2 having the amino acid sequence setforth in SEQ ID NO: 13 or 14; and a heavy chain variable region CDR3having the amino acid sequence set forth in SEQ ID NO: 15 or 70; and b)a light chain variable region CDR1 having the amino acid sequence setforth in SEQ ID NO: 23; a light chain variable region CDR2 having theamino acid sequence set forth in SEQ ID NO: 24; and a light chainvariable region CDR3 having the amino acid sequence set forth in SEQ IDNO:
 25. 16. An antibody or antigen-binding fragment thereof, comprising:a) a heavy chain variable region CDR1 having the amino acid sequence setforth in SEQ ID NO: 5; a heavy chain variable region CDR2 having theamino acid sequence set forth in SEQ ID NO: 8; and a heavy chainvariable region CDR3 having the amino acid sequence set forth in SEQ IDNO: 11; and b) a light chain variable region CDR1 having the amino acidsequence set forth in SEQ ID NO: 20; a light chain variable region CDR2having the amino acid sequence set forth in SEQ ID NO: 21; and a lightchain variable region CDR3 having the amino acid sequence set forth inSEQ ID NO:
 22. 17. The antibody or antigen-binding fragment thereof ofclaim 16, comprising: a) an immunoglobulin heavy chain variable regionhaving the amino acid sequence set forth in SEQ ID NO: 34; and b) animmunoglobulin light chain variable region having the amino acidsequence set forth in SEQ ID NO:
 35. 18. The antibody or antigen-bindingfragment thereof of claim 17, wherein Xaa, the second residue from theN-terminus of SEQ ID NO: 34, is Val.
 19. A polypeptide comprising theheavy chain variable region and the light chain variable regionsequences of claim
 17. 20. The antibody or antigen-binding fragmentthereof of claim 16, comprising: a) an immunoglobulin heavy chain havingthe amino acid sequence set forth in SEQ ID NO: 54; and b) animmunoglobulin light chain variable region having the amino acidsequence set forth in SEQ ID NO:
 35. 21. The antibody or antigen-bindingfragment thereof of claim 20, wherein Xaa, the second residue from theN-terminus of SEQ ID NO: 54, is Val.
 22. The antibody or antigen-bindingfragment thereof of claim 16, comprising: a) an immunoglobulin heavychain variable region having the amino acid sequence set forth in SEQ IDNO: 34; and b) an immunoglobulin light chain having the amino acidsequence set forth in SEQ ID NO:
 51. 23. The antibody or antigen-bindingfragment thereof of claim 22, wherein Xaa, the second residue from theN-terminus of SEQ ID NO: 34, is Val.
 24. The antibody or antigen-bindingfragment thereof of claim 16, wherein the antibody comprises: a) animmunoglobulin heavy chain having the amino acid sequence set forth inSEQ ID NO: 54; and b) an immunoglobulin light chain having the aminoacid sequence set forth in SEQ ID NO:
 51. 25. The antibody orantigen-binding fragment thereof of claim 24, wherein Xaa, the secondresidue from the N-terminus of SEQ ID NO: 54, is Val.
 26. A cellproducing the antibody or antigen-binding fragment thereof of claim 16.27. A method of producing the antibody or antigen-binding fragmentthereof of claim 16, comprising: (a) culturing a cell producing saidantibody or antigen-binding fragment thereof; and, (b) isolating saidantibody, antigen-binding fragment thereof, or polypeptide from saidcultured cell.
 28. A pharmaceutical composition comprising the antibodyor antigen-binding fragment thereof of claim 16, and a pharmaceuticallyacceptable carrier.